Tbx3 gene and methods of using it

ABSTRACT

The present invention relates to all facets of a novel polynucleotide, Tbx3-pr408, the polypeptides it encodes, antibodies and specific binding partners thereto, and their applications to research, diagnosis, drug discovery, therapy, clinical medicine, forensic science and medicine, etc. The polynucleotides useful in variety of ways, including, but not limited to, as molecular markers, as drug targets, and for detecting, diagnosing, staging, monitoring, prognosticating, preventing or treating, determining predisposition to, etc., diseases and conditions, such as mammary-ulnar syndrome and other developmental disorders.

DESCRIPTION OF THE DRAWINGS

[0001] SEQ ID NOS 1 and 2 show the nucleotide and amino acid sequencesof Tbx3-pr408. The polynucleotides are human cDNAs.

[0002]FIG. 1 shows the amino acid comparisons between Tbx3-pr408 (SEQ IDNO 2), NM_(—)005996 (SEQ ID NO 3), XM_(—)016321 (SEQ ID NO 4), andNM_(—)016569 (SEQ ID NO 5).

DESCRIPTION OF THE INVENTION

[0003] The present invention relates to all facets of Tbx3-pr408,polypeptides encoded by it, antibodies and specific binding partnersthereto, and their applications to research, diagnosis, drug discovery,therapy, clinical medicine, forensic science and medicine, etc.Tbx3-pr408 polynucleotides, polypeptides, antibodies, etc., are usefulin variety of ways, including, but not limited to, as a molecularmarkers, as drug targets, and for detecting, diagnosing, staging,monitoring, prognosticating, preventing or treating, determiningpredisposition to, etc., diseases and conditions, such as mammary-ulnarsyndrome and other developmental disorders associated with it, as wellas conditions associated with the adult tissues in which Tbx3-pr408 isexpressed. The present invention also relates to methods of using thepolynucleotides and related products (proteins, antibodies, etc.) inbusiness and computer-related methods, e.g., advertising, displaying,offering, selling, etc., such products for sale, commercial use,licensing, etc.

[0004] Tbx3-pr408 codes for a polypeptide having 723 amino acids. It isa transcription factor. The nucleotide and amino acid sequences ofTbx3-pr408 are shown in SEQ ID NOS 1 and 2. A T box domain, havingDNA-binding activity, is located at about amino acid positions 102-290.

[0005] Tbx3-pr408 is a transcript of the Tbx3 gene. See, e.g., He etal., Proc. Natl. Acad. Sci., 96:10212-10217, 1999. It maps tochromosomal position 12q24.1. Mutations in this gene cause ulnar-mammarysyndrome, a disease associated with anomalies in limb, apocrine andgenital development. Bamshad et al., Nat. Genet., 16:311-316, 1997. FIG.1 shows the alignment between Tbx3-pr408 and previously knowntranscripts (NM_(—)005996, XM_(—)016321, and NM_(—)016569) of the Tbx3gene. As indicated in FIG. 1, Pr408 is decidedly different. In additionto other differences, XM_(—)016321, and NM_(—)016569 contain a 20 aminoacid insertion in the T box domain between amino acids 220 and 221 ofTbx3-pr408. This insertion appears to alter the DNA-binding activity ofthe T box. NM_(—)005996 is at least one amino acid shorter thanTbx3-pr408 and contains different sequence at amino acid positions596-608.

[0006] Tbx3-pr408 has several different biological activities,including, e.g., transcription modulatory activity, and DNA-bindingactivity. Its activity can be determined routinely. For instance, itstranscriptional regulatory activity can be assessed in transcriptionreporter assays in which Tbx3-pr408, or fragments thereof, can be fusedDNA-binding elements (e.g., LexA or Gal4) and/or transactivators, thatare used to modulate expression of reporter genes, e.g., as described inHe et al., Proc. Natl. Acad. Sci., 96:10212-10217, 1999. By the phrase“transcription regulatory activity,” it is meant that the polypeptidemodulates transcription of a gene. This modulatory activity can beactivation or repression (e.g., He et al. describe repression for a Tbx3domain). DNA-binding activity can be determined using gel-shift assays.

[0007] Consistent with the phenotype of mammary-ulnar syndrome,Tbx3-pr408 is expressed in the testes and prostate. Additionally, it isexpressed at high levels in the adrenal gland, heart, lung, and muscle.Lower levels are observed in other tissues, as well. Tissues were alsoassayed for the presence of the Tbx3 transcript having the insertionbetween amino acids 220 and 221. Both this transcript and Tbx3-pr408were expressed in most tissues, however, Tbx3-pr408 was more abundant.Heart expression is particularly striking since mutations in the Tbx5gene, adjacent to the Tbx3 gene on chromosome 12, is associated withcardiac anomalies, suggesting that both genes may play a role in heartdevelopment.

[0008] In addition to its association with ulnar-mammary syndrome,Tbx3-pr408 may also have a role in other developmental disorders,including, e.g., spinal muscular atrophy (congenital nonprogressive, oflower limbs), type C brachydactyly, B-cell non-Hodgkin lymphoma, andscapuloperoneal spinal muscular atrophy (New England type). Nucleicacids of the present invention can be used as linkage markers,diagnostic targets, therapeutic targets, for any of the mentioneddisorders, as well as any disorders or genes mapping in proximity to it.Nucleic acids

[0009] A mammalian polynucleotide, or fragment thereof, of the presentinvention is a polynucleotide having a nucleotide sequence obtainablefrom a natural source. It therefore includes naturally-occurring normal,naturally-occurring mutant, and naturally-occurring polymorphic alleles(e.g., SNPs), differentially-spliced transcripts, splice-variants, etc.By the term “naturally-occurring,” it is meant that the polynucleotideis obtainable from a natural source, e.g., animal tissue and cells, bodyfluids, tissue culture cells, forensic samples. Natural sources include,e.g., living cells obtained from tissues and whole organisms, tumors,cultured cell lines, including primary and immortalized cell lines.Naturally-occurring mutations can include deletions (e.g., a truncatedamino- or carboxy-terminus), substitutions, inversions, or additions ofnucleotide sequence. These genes can be detected and isolated bypolynucleotide hybridization according to methods which one skilled inthe art would know, e.g., as discussed below.

[0010] A polynucleotide according to the present invention can beobtained from a variety of different sources. It can be obtained fromDNA or RNA, such as polyadenylated mRNA or total RNA, e.g., isolatedfrom tissues, cells, or whole organism. The polynucleotide can beobtained directly from DNA or RNA, from a cDNA library, from a genomiclibrary, etc. The polynucleotide can be obtained from a cell or tissue(e.g., from an embryonic or adult tissues) at a particular stage ofdevelopment, having a desired genotype, phenotype, disease status, etc.These sequences can be obtained by any suitable method, e.g., using apartial sequence as a probe to select a full-length cDNA from a librarycontaining full-length inserts. A polynucleotide which “codes withoutinterruption” refers to a polynucleotide having a continuous openreading frame (“ORF”) as compared to an ORF which is interrupted byintrons or other noncoding sequences.

[0011] Polynucleotides and polypeptides (including any part ofTbx3-pr408) can be excluded as compositions from the present inventionif, e.g., listed in a publicly available databases on the day thisapplication was filed and/or disclosed in a patent application having anearlier filing or priority date than this application and/or conceivedand/or reduced to practice earlier than a polynucleotide in thisapplication.

[0012] As described herein, the phrase “an isolated polynucleotide whichis SEQ ID NO,” or “an isolated polynucleotide which is selected from SEQID NO,” refers to an isolated nucleic acid molecule from which therecited sequence was derived (e.g., a cDNA derived from mRNA; cDNAderived from genomic DNA). Because of sequencing errors, typographicalerrors, etc., the actual naturally-occurring sequence may differ from aSEQ ID listed herein. Thus, the phrase indicates the specific moleculefrom which the sequence was derived, rather than a molecule having thatexact recited nucleotide sequence, analogously to how a culturedepository number refers to a specific cloned fragment in a cryotube.

[0013] As explained in more detail below, a polynucleotide sequence ofthe invention can contain the complete sequence as shown in SEQ ID NO 1,degenerate sequences thereof, anti-sense, muteins thereof, genescomprising said sequences, full-length cDNAs comprising said sequences,complete genomic sequences, fragments thereof, homologs, primers,nucleic acid molecules which hybridize thereto, derivatives thereof,etc.

[0014] Constructs

[0015] A polynucleotide of the present invention can comprise additionalpolynucleotide sequences, e.g., sequences to enhance expression,detection, uptake, cataloging, tagging, etc. A polynucleotide caninclude only coding sequence; a coding sequence and additionalnon-naturally occurring or heterologous coding sequence (e.g., sequencescoding for leader, signal, secretory, targeting, enzymatic, fluorescent,antibiotic resistance, and other functional or diagnostic peptides);coding sequences and non-coding sequences, e.g., untranslated sequencesat either a 5′ or 3′ end, or dispersed in the coding sequence, e.g.,introns.

[0016] A polynucleotide according to the present invention also cancomprise an expression control sequence operably linked to apolynucleotide as described above. The phrase “expression controlsequence” means a polynucleotide sequence that regulates expression of apolypeptide coded for by a polynucleotide to which it is functionally(“operably”) linked. Expression can be regulated at the level of themRNA or polypeptide. Thus, the expression control sequence includesmRNA-related elements and protein-related elements. Such elementsinclude promoters, enhancers (viral or cellular), ribosome bindingsequences, transcriptional terminators, etc. An expression controlsequence is operably linked to a nucleotide coding sequence when theexpression control sequence is positioned in such a manner to effect orachieve expression of the coding sequence. For example, when a promoteris operably linked 5′ to a coding sequence, expression of the codingsequence is driven by the promoter. Expression control sequences caninclude an initiation codon and additional nucleotides to place apartial nucleotide sequence of the present invention in-frame in orderto produce a polypeptide (e.g., pET vectors from Promega have beendesigned to permit a molecule to be inserted into all three readingframes to identify the one that results in polypeptide expression).Expression control sequences can be heterologous or endogenous to thenormal gene.

[0017] A polynucleotide of the present invention can also comprisenucleic acid vector sequences, e.g., for cloning, expression,amplification, selection, etc. Any effective vector can be used. Avector is, e.g., a polynucleotide molecule which can replicateautonomously in a host cell, e.g., containing an origin of replication.Vectors can be useful to perform manipulations, to propagate, and/orobtain large quantities of the recombinant molecule in a desired host. Askilled worker can select a vector depending on the purpose desired,e.g., to propagate the recombinant molecule in bacteria, yeast, insect,or mammalian cells. The following vectors are provided by way ofexample. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10,Phagescript, phiX174, pBK Phagemid, pNH8A, pNH16a, pNH18Z, pNH46A(Stratagene); Bluescript KS+II (Stratagene); ptrc99a, pKK223-3,pKK233-3, pDR54 0, pRIT5 (Pharmacia). Eukaryotic: PWLNEO, pSV2CAT,pOG44, pXT1, pSG (Stratagene), pSVK3, PBPV, PMSG, pSVL (Pharmacia),pCR2. 1/TOPO, pCRII/TOPO, pCR4/TOPO, pTrcHisB, pCMV6-XL4, etc. However,any other vector, e.g., plasmids, viruses, or parts thereof, may be usedas long as they are replicable and viable in the desired host. Thevector can also comprise sequences which enable it to replicate in thehost whose genome is to be modified.

[0018] Hybridization

[0019] Polynucleotide hybridization, as discussed in more detail below,is useful in a variety of applications, including, in gene detectionmethods, for identifying mutations, for making mutations, to identifyhomologs in the same and different species, to identify related membersof the same gene family, in diagnostic and prognostic assays, intherapeutic applications (e.g., where an antisense polynucleotide isused to inhibit expression), etc.

[0020] The ability of two single-stranded polynucleotide preparations tohybridize together is a measure of their nucleotide sequencecomplementarity, e.g., base-pairing between nucleotides, such as A-T,G-C, etc. The invention thus also relates to polynucleotides, and theircomplements, which hybridize to a polynucleotide comprising a nucleotidesequence as set forth in SEQ ID NO 1 and genomic sequences thereof. Anucleotide sequence hybridizing to the latter sequence will have acomplementary polynucleotide strand, or act as a template for one in thepresence of a polymerase (i.e., an appropriate polynucleotidesynthesizing enzyme). The present invention includes both strands ofpolynucleotide, e.g., a sense strand and an anti-sense strand.

[0021] Hybridization conditions can be chosen to select polynucleotideswhich have a desired amount of nucleotide complementarity with thenucleotide sequences set forth in SEQ ID NO 1 and genomic sequencesthereof. A polynucleotide capable of hybridizing to such sequence,preferably, possesses, e.g., about 70%, 75%, 80%, 85%, 87%, 90%, 92%,95%, 97%, 99%, or 100% complementarity, between the sequences. Thepresent invention particularly relates to polynucleotide sequences whichhybridize to the nucleotide sequences set forth in SEQ ID NO 1 orgenomic sequences thereof, under low or high stringency conditions.These conditions can be used, e.g., to select corresponding homologs innon-human species.

[0022] Polynucleotides which hybridize to polynucleotides of the presentinvention can be selected in various ways. Filter-type blots (i.e.,matrices containing polynucleotide, such as nitrocellulose), glasschips, and other matrices and substrates comprising polynucleotides(short or long) of interest, can be incubated in a prehybridizationsolution (e.g., 6×SSC, 0.5% SDS, 100 μg/ml denatured salmon sperm DNA,5×Denhardt's solution, and 50% formamide), at 22-68° C., overnight, andthen hybridized with a detectable polynucleotide probe under conditionsappropriate to achieve the desired stringency. In general, when highhomology or sequence identity is desired, a high temperature can be used(e.g., 65° C.). As the homology drops, lower washing temperatures areused. For salt concentrations, the lower the salt concentration, thehigher the stringency. The length of the probe is another consideration.Very short probes (e.g., less than 100 base pairs) are washed at lowertemperatures, even if the homology is high. With short probes, formamidecan be omitted. See, e.g., Current Protocols in Molecular Biology,Chapter 6, Screening of Recombinant Libraries; Sambrook et al.,Molecular Cloning, 1989, Chapter 9.

[0023] For instance, high stringency conditions can be achieved byincubating the blot overnight (e.g., at least 12 hours) with a longpolynucleotide probe in a hybridization solution containing, e.g., about5×SSC, 0.5% SDS, 100 μg/ml denatured salmon sperm DNA and 50% formamide,at 42° C. Blots can be washed at high stringency conditions that allow,e.g., for less than 5% bp mismatch (e.g., wash twice in 0.1% SSC and0.1% SDS for 30 min at 65° C.), i.e., selecting sequences having 95% orgreater sequence identity.

[0024] Other non-limiting examples of high stringency conditionsincludes a final wash at 65° C. in aqueous buffer containing 30 mM NaCland 0.5% SDS. Another example of high stringent conditions ishybridization in 7% SDS, 0.5 M NaPO₄, pH 7, 1 mM EDTA at 50° C., e.g.,overnight, followed by one or more washes with a 1% SDS solution at 42°C. Whereas high stringency washes can allow for less than 5% mismatch,reduced or low stringency conditions can permit up to 20% nucleotidemismatch. Hybridization at low stringency can be accomplished as above,but using lower formamide conditions, lower temperatures and/or lowersalt concentrations, as well as longer periods of incubation time.

[0025] Hybridization can also be based on a calculation of meltingtemperature (Tm) of the hybrid formed between the probe and its target,as described in Sambrook et al., Generally, the temperature Tm at whicha short oligonucleotide (containing 18 nucleotides or fewer) will meltfrom its target sequence is given by the following equation: Tm=(numberof A's and T's)×2° C.+(number of C's and G's)×4° C. For longermolecules, Tm=81.5+16.6 log₁₀[Na⁺]+0.41(% GC)−600/N where [Na⁺] is themolar concentration of sodium ions, % GC is the percentage of GC basepairs in the probe, and N is the length. Hybridization can be carriedout at several degrees below this temperature to ensure that the probeand target can hybridize. Mismatches can be allowed for by lowering thetemperature even further.

[0026] Stringent conditions can be selected to isolate sequences, andtheir complements, which have, e.g., at least about 90%, 95%, or 97%,nucleotide complementarity between the probe (e.g., a shortpolynucleotide of SEQ ID NO 1 or genomic sequences thereof) and a targetpolynucleotide.

[0027] Other homologs of polynucleotides of the present invention can beobtained from mammalian and non-mammalian sources according to variousmethods. For example, hybridization with a polynucleotide can beemployed to select homologs, e.g., as described in Sambrook et al.,Molecular Cloning, Chapter 11, 1989. Such homologs can have varyingamounts of nucleotide and amino acid sequence identity and similarity tosuch polynucleotides of the present invention. Mammalian organismsinclude, e.g., mice, rats, monkeys, pigs, cows, etc. Non-mammalianorganisms include, e.g., vertebrates, invertebrates, zebra fish,chicken, Drosophila, C. elegans, Xenopus, yeast such as S. pombe, S.cerevisiae, roundworms, prokaryotes, plants, Arabidopsis, artemia,viruses, etc. The degree of nucleotide sequence identity between humanand mouse can be about, e.g. 70% or more, 85% or more for open readingframes, etc.

[0028] Alignment

[0029] Alignments can be accomplished by using any effective algorithm.For pairwise alignments of DNA sequences, the methods described byWilbur-Lipman (e.g., Wilbur and Lipman, Proc. Natl. Acad. Sci.,80:726-730, 1983) or Martinez/Needleman-Wunsch (e.g., Martinez, NucleicAcid Res., 11:4629-4634, 1983) can be used. For instance, if theMartinez/Needleman-Wunsch DNA alignment is applied, the minimum matchcan be set at 9, gap penalty at 1.10, and gap length penalty at 0.33.The results can be calculated as a similarity index, equal to the sum ofthe matching residues divided by the sum of all residues and gapcharacters, and then multiplied by 100 to express as a percent.Similarity index for related genes at the nucleotide level in accordancewith the present invention can be greater than 70%, 80%, 85%, 90%, 95%,99%, or more. Pairs of protein sequences can be aligned by theLipman-Pearson method (e.g., Lipman and Pearson, Science, 227:1435-1441,1985) with k-tuple set at 2, gap penalty set at 4, and gap lengthpenalty set at 12. Results can be expressed as percent similarity index,where related genes at the amino acid level in accordance with thepresent invention can be greater than 65%, 70%, 75%, 80%, 85%, 90%, 95%,99%, or more. Various commercial and free sources of alignment programsare available, e.g., MegAlign by DNA Star, BLAST (National Center forBiotechnology Information), BCM (Baylor College of Medicine) Launcher,etc.

[0030] Percent sequence identity can also be determined by otherconventional methods, e.g., as described in Altschul et al., Bull. Math.Bio. 48: 603-616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci.USA 89:10915-10919, 1992.

[0031] Specific Polynucleotide Probes

[0032] A polynucleotide of the present invention can comprise anycontinuous nucleotide sequence of SEQ ID NO 1, sequences which sharesequence identity thereto, or complements thereof. The term “probe”refers to any substance that can be used to detect, identify, isolate,etc., another substance. A polynucleotide probe is comprised of nucleicacid can be used to detect, identify, etc., other nucleic acids, such asDNA and RNA. Useful probes, include, e.g., probes on either side ofamino acid positions 220-221 (i.e., where NM_(—)005996 and XM_(—)016321have insertions), e.g., a probe selected from nucleotide positions1112-1183 and from nucleotide positions 1184-1255) to determine whetherthe insertion is present or not.

[0033] These polynucleotides can be of any desired size that iseffective to achieve the specificity desired. For example, a probe canbe from about 7 or 8 nucleotides to several thousand nucleotides,depending upon its use and purpose. For instance, a probe used as aprimer PCR can be shorter than a probe used in an ordered array ofpolynucleotide probes. Probe sizes vary, and the invention is notlimited in any way by their size, e.g., probes can be from about 7-2000nucleotides, 7-1000, 8-700, 8-600, 8-500, 8-400, 8-300, 8-150, 8-100,8-75, 7-50, 10-25, 14-16, at least about 8, at least about 10, at leastabout 15, at least about 25, etc. The polynucleotides can havenon-naturally-occurring nucleotides, e.g., inosine, AZT, 3TC, etc. Thepolynucleotides can have 100% sequence identity or complementarity to asequence of SEQ ID NO 1, or it can have mismatches or nucleotidesubstitutions, e.g., 1, 2, 3, 4, or 5 substitutions. The probes can besingle-stranded or double-stranded.

[0034] In accordance with the present invention, a polynucleotide can bepresent in a kit, where the kit includes, e.g., one or morepolynucleotides, a desired buffer (e.g., phosphate, tris, etc.),detection compositions, RNA or cDNA from different tissues to be used ascontrols, libraries, etc. The polynucleotide can be labeled orunlabeled, with radioactive or non-radioactive labels as known in theart. Kits can comprise one or more pairs of polynucleotides foramplifying nucleic acids specific for Tbx3-pr408, e.g., comprising aforward and reverse primer effective in PCR. These include both senseand anti-sense orientations. For instance, in PCR-based methods (such asRT-PCR), a pair of primers are typically used, one having a sensesequence and the other having an antisense sequence.

[0035] Another aspect of the present invention is a nucleotide sequencethat is specific to, or for, a selective polynucleotide. The phrases“specific for” or “specific to” a polynucleotide have a functionalmeaning that the polynucleotide can be used to identify the presence ofone or more target genes in a sample. It is specific in the sense thatit can be used to detect polynucleotides above background noise(“non-specific binding”). A specific sequence is a defined order ofnucleotides which occurs in the polynucleotide, e.g., in the nucleotidesequences of SEQ ID NO 1. A probe or mixture of probes can comprise asequence or sequences that are specific to a plurality of targetsequences, e.g., where the sequence is a consensus sequence, afunctional domain, etc., e.g., capable of recognizing a family ofrelated genes. Such sequences can be used as probes in any of themethods described herein or incorporated by reference. Both sense andantisense nucleotide sequences are included. A specific polynucleotideaccording to the present invention can be determined routinely.

[0036] A polynucleotide comprising a specific sequence can be used as ahybridization probe to identify the presence of, e.g., human or mousepolynucleotide, in a sample comprising a mixture of polynucleotides,e.g., on a Northern blot. Hybridization can be performed under highstringent conditions (see, above) to select polynucleotides (and theircomplements which can contain the coding sequence) having at least 90%,95%, 99%, etc., identity (i.e., complementarity) to the probe, but lessstringent conditions can also be used. A specific polynucleotidesequence can also be fused in-frame, at either its 5′ or 3′ end, tovarious nucleotide sequences as mentioned throughout the patent,including coding sequences for enzymes, detectable markers, GFP, etc,expression control sequences, etc.

[0037] A polynucleotide probe, especially one that is specific to apolynucleotide of the present invention, can be used in gene detectionand hybridization methods as already described. In one embodiment, aspecific polynucleotide probe can be used to detect whether a particulartissue or cell-type is present in a target sample. To carry out such amethod, a selective polynucleotide can be chosen which is characteristicof the desired target tissue. Such polynucleotide is preferably chosenso that it is expressed or displayed in the target tissue, but not inother tissues which are present in the sample. For instance, ifdetection of prostate is desired, it may not matter whether theselective polynucleotide is expressed in other tissues, as long as it isnot expressed in cells normally present in blood, e.g., peripheral bloodmononuclear cells. Starting from the selective polynucleotide, aspecific polynucleotide probe can be designed which hybridizes (ifhybridization is the basis of the assay) under the hybridizationconditions to the selective polynucleotide, whereby the presence of theselective polynucleotide can be determined.

[0038] Probes which are specific for polynucleotides of the presentinvention can also be prepared using involve transcription-basedsystems, e.g., incorporating an RNA polymerase promoter into a selectivepolynucleotide of the present invention, and then transcribinganti-sense RNA using the polynucleotide as a template. See, e.g., U.S.Pat. No. 5,545,522.

[0039] Polynucleotide Composition

[0040] A polynucleotide according to the present invention can comprise,e.g., DNA, RNA, synthetic polynucleotide, peptide polynucleotide,modified nucleotides, dsDNA, ssDNA, ssRNA, dsRNA, and mixtures thereof.A polynucleotide can be single- or double-stranded, triplex, DNA:RNA,duplexes, comprise hairpins, and other secondary structures, etc.Nucleotides comprising a polynucleotide can be joined via various knownlinkages, e.g., ester, sulfamate, sulfamide, phosphorothioate,phosphoramidate, methylphosphonate, carbamate, etc., depending on thedesired purpose, e.g., resistance to nucleases, such as RNAse H,improved in vivo stability, etc. See, e.g., U.S. Pat. No. 5,378,825. Anydesired nucleotide or nucleotide analog can be incorporated, e.g.,6-mercaptoguanine, 8-oxo-guanine, etc.

[0041] Various modifications can be made to the polynucleotides, such asattaching detectable markers (avidin, biotin, radioactive elements,fluorescent tags and dyes, energy transfer labels, energy-emittinglabels, binding partners, etc.) or moieties which improve hybridization,detection, and/or stability. The polynucleotides can also be attached tosolid supports, e.g., nitrocellulose, magnetic or paramagneticmicrospheres (e.g., as described in U.S. Pat. No. 5,411,863; U.S. Pat.No. 5,543,289; for instance, comprising ferromagnetic, supermagnetic,paramagnetic, superparamagnetic, iron oxide and polysaccharide), nylon,agarose, diazotized cellulose, latex solid microspheres,polyacrylamides, etc., according to a desired method. See, e.g., U.S.Pat. Nos. 5,470,967, 5,476,925, and 5,478,893.

[0042] Polynucleotide according to the present invention can be labeledaccording to any desired method. The polynucleotide can be labeled usingradioactive tracers such as ³²P, ³⁵S, ³H, or ¹⁴C, to mention somecommonly used tracers. The radioactive labeling can be carried outaccording to any method, such as, for example, terminal labeling at the3′ or 5′ end using a radio labeled nucleotide, polynucleotide kinase(with or without dephosphorylation with a phosphatase) or a ligase(depending on the end to be labeled). A non-radioactive labeling canalso be used, combining a polynucleotide of the present invention withresidues having immunological properties (antigens, haptens), a specificaffinity for certain reagents (ligands), properties enabling detectableenzyme reactions to be completed (enzymes or coenzymes, enzymesubstrates, or other substances involved in an enzymatic reaction), orcharacteristic physical properties, such as fluorescence or the emissionor absorption of light at a desired wavelength, etc.

[0043] Nucleic Acid Detection Methods

[0044] Another aspect of the present invention relates to methods andprocesses for detecting Tbx3-pr408. Detection methods have a variety ofapplications, including for diagnostic, prognostic, forensic, andresearch applications. To accomplish gene detection, a polynucleotide inaccordance with the present invention can be used as a “probe.” The term“probe” or “polynucleotide probe” has its customary meaning in the art,e.g., a polynucleotide which is effective to identify (e.g., byhybridization), when used in an appropriate process, the presence of atarget polynucleotide to which it is designed. Identification caninvolve simply determining presence or absence, or it can bequantitative, e.g., in assessing amounts of a gene or gene transcriptpresent in a sample. Probes can be useful in a variety of ways, such asfor diagnostic purposes, to identify homologs, and to detect,quantitate, or isolate a polynucleotide of the present invention in atest sample.

[0045] Assays can be utilized which permit quantification and/orpresence/absence detection of a target nucleic acid in a sample. Assayscan be performed at the single-cell level, or in a sample comprisingmany cells, where the assay is “averaging” expression over the entirecollection of cells and tissue present in the sample. Any suitable assayformat can be used, including, but not limited to, e.g., Southern blotanalysis, Northern blot analysis, polymerase chain reaction (“PCR”)(e.g., Saiki et al., Science, 241:53, 1988; U.S. Pat. Nos. 4,683,195,4,683,202, and 6,040,166; PCR Protocols: A Guide to Methods andApplications, Innis et al., eds., Academic Press, New York, 1990),reverse transcriptase polymerase chain reaction (“RT-PCR”), anchoredPCR, rapid amplification of cDNA ends (“RACE”) (e.g., Schaefer in GeneCloning and Analysis: Current Innovations, Pages 99-115, 1997), ligasechain reaction (“LCR”) (EP 320 308), one-sided PCR (Ohara et al., Proc.Natl. Acad. Sci., 86:5673-5677, 1989), indexing methods (e.g., U.S. Pat.No. 5,508,169), in situ hybridization, differential display (e.g., Lianget al., Nucl. Acid. Res., 21:3269-3275, 1993; U.S. Pat. Nos. 5,262,311,5,599,672 and 5,965,409; WO97/18454; Prashar and Weissman, Proc. Natl.Acad. Sci., 93:659-663, and U.S. Pat. Nos. 6,010,850 and 5,712,126;Welsh et al., Nucleic Acid Res., 20:4965-4970, 1992, and U.S. Pat. No.5,487,985) and other RNA fingerprinting techniques, nucleic acidsequence based amplification (“NASBA”) and other transcription basedamplification systems (e.g., U.S. Pat. Nos. 5,409,818 and 5,554,527; WO88/10315), polynucleotide arrays (e.g., U.S. Pat. Nos. 5,143,854,5,424,186; 5,700,637, 5,874,219, and 6,054,270; PCT WO 92/10092; PCT WO90/15070), Qbeta Replicase (PCT/US87/00880), Strand DisplacementAmplification (“SDA”), Repair Chain Reaction (“RCR”), nucleaseprotection assays, subtraction-based methods, Rapid-Scan™, etc.Additional useful methods include, but are not limited to, e.g.,template-based amplification methods, competitive PCR (e.g., U.S. Pat.No. 5,747,251), redox-based assays (e.g., U.S. Pat. No. 5,871,918),Taqman-based assays (e.g., Holland et al., Proc. Natl. Acad, Sci.,88:7276-7280, 1991; U.S. Pat. Nos. 5,210,015 and 5,994,063), real-timefluorescence-based monitoring (e.g., U.S. Pat. No. 5,928,907), molecularenergy transfer labels (e.g., U.S. Pat. Nos. 5,348,853, 5,532,129,5,565,322, 6,030,787, and 6,117,635; Tyagi and Kramer, Nature Biotech.,14:303-309, 1996). Any method suitable for single cell analysis of geneor protein expression can be used, including in situ hybridization,immunocytochemistry, MACS, FACS, flow cytometry, etc. For single cellassays, expression products can be measured using antibodies, PCR, orother types of nucleic acid amplification (e.g., Brady et al., MethodsMol. & Cell. Biol. 2, 17-25, 1990; Eberwine et al., 1992, Proc. Natl.Acad. Sci., 89, 3010-3014, 1992; U.S. Pat. No. 5,723,290). These andother methods can be carried out conventionally, e.g., as described inthe mentioned publications.

[0046] Many of such methods may require that the polynucleotide islabeled, or comprises a particular nucleotide type useful for detection.The present invention includes such modified polynucleotides that arenecessary to carry out such methods. Thus, polynucleotides can be DNA,RNA, DNA:RNA hybrids, PNA, etc., and can comprise any modification orsubstituent which is effective to achieve detection.

[0047] Detection can be desirable for a variety of different purposes,including research, diagnostic, prognostic, and forensic. For diagnosticpurposes, it may be desirable to identify the presence or quantity of apolynucleotide sequence in a sample, where the sample is obtained fromtissue, cells, body fluids, etc. In a preferred method as described inmore detail below, the present invention relates to a method ofdetecting a polynucleotide comprising, contacting a targetpolynucleotide in a test sample with a polynucleotide probe underconditions effective to achieve hybridization between the target andprobe; and detecting hybridization.

[0048] Any test sample in which it is desired to identify apolynucleotide or polypeptide thereof can be used, including, e.g.,blood, urine, saliva, stool (for extracting nucleic acid, see, e.g.,U.S. Pat. No. 6,177,251), swabs comprising tissue, biopsied tissue,tissue sections, cultured cells, etc.

[0049] Detection can be accomplished in combination with polynucleotideprobes for other genes, e.g., genes which are expressed in other diseasestates, tissues, cells, such as brain, heart, kidney, spleen, thymus,liver, stomach, small intestine, colon, muscle, lung, testis, placenta,pituitary, thyroid, skin, adrenal gland, pancreas, salivary gland,uterus, ovary, prostate gland, peripheral blood cells (T-cells,lymphocytes, etc.), embryo, normal breast fat, adult and embryonic stemcells, specific cell-types, such as endothelial, epithelial, myocytes,adipose, luminal epithelial, basoepithelial, myoepithelial, stromalcells, etc.

[0050] Polynucleotides can be used in wide range of methods andcompositions, including for detecting, diagnosing, staging, grading,assessing, prognosticating, etc. diseases and disorders associated withTbx3-pr408, for monitoring or assessing therapeutic and/or preventativemeasures, in ordered arrays, etc. Any method of detecting genes andpolynucleotides of SEQ ID NO 1 can be used; certainly, the presentinvention is not to be limited how such methods are implemented.

[0051] Along these lines, the present invention relates to methods ofdetecting Tbx3-pr408 in a sample comprising nucleic acid. Such methodscan comprise one or more the following steps in any effective order,e.g., contacting said sample with a polynucleotide probe underconditions effective for said probe to hybridize specifically to nucleicacid in said sample, and detecting the presence or absence of probehybridized to nucleic acid in said sample, wherein said probe is apolynucleotide which is SEQ ID NO 1, a polynucleotide having, e.g.,about 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity thereto,effective or specific fragments thereof, or complements thereto. Thedetection method can be applied to any sample, e.g., cultured primary,secondary, or established cell lines, tissue biopsy, blood, urine,stool, cerebral spinal fluid, and other bodily fluids, for any purpose.

[0052] Contacting the sample with probe can be carried out by anyeffective means in any effective environment. It can be accomplished ina solid, liquid, frozen, gaseous, amorphous, solidified, coagulated,colloid, etc., mixtures thereof, matrix. For instance, a probe in anaqueous medium can be contacted with a sample which is also in anaqueous medium, or which is affixed to a solid matrix, or vice-versa.

[0053] Generally, as used throughout the specification, the term“effective conditions”means, e.g., the particular milieu in which thedesired effect is achieved. Such a milieu, includes, e.g., appropriatebuffers, oxidizing agents, reducing agents, pH, co-factors, temperature,ion concentrations, suitable age and/or stage of cell (such as, inparticular part of the cell cycle, or at a particular stage whereparticular genes are being expressed) where cells are being used,culture conditions (including substrate, oxygen, carbon dioxide, etc.).When hybridization is the chosen means of achieving detection, the probeand sample can be combined such that the resulting conditions arefunctional for said probe to hybridize specifically to nucleic acid insaid sample.

[0054] The phrase “hybridize specifically” indicates that thehybridization between single-stranded polynucleotides is based onnucleotide sequence complementarity. The effective conditions areselected such that the probe hybridizes to a preselected and/or definitetarget nucleic acid in the sample. For instance, if detection of apolynucleotide set forth in SEQ ID NO 1 is desired, a probe can beselected which can hybridize to such target gene under high stringentconditions, without significant hybridization to other genes in thesample. To detect homologs of a polynucleotide set forth in SEQ ID NO 1,the effective hybridization conditions can be less stringent, and/or theprobe can comprise codon degeneracy, such that a homolog is detected inthe sample.

[0055] As already mentioned, the methods can be carried out by anyeffective process, e.g., by Northern blot analysis, polymerase chainreaction (PCR), reverse transcriptase PCR, RACE PCR, in situhybridization, etc., as indicated above. When PCR based techniques areused, two or more probes are generally used. One probe can be specificfor a defined sequence which is characteristic of a selectivepolynucleotide, but the other probe can be specific for the selectivepolynucleotide, or specific for a more general sequence, e.g., asequence such as polyA which is characteristic of mRNA, a sequence whichis specific for a promoter, ribosome binding site, or othertranscriptional features, a consensus sequence (e.g., representing afunctional domain). For the former aspects, 5′ and 3′ probes (e.g.,polyA, Kozak, etc.) are preferred which are capable of specificallyhybridizing to the ends of transcripts. When PCR is utilized, the probescan also be referred to as “primers” in that they can prime a DNApolymerase reaction.

[0056] In addition to testing for the presence or absence ofpolynucleotides, the present invention also relates to determining theamounts at which polynucleotides of the present invention are expressedin sample and determining the differential expression of suchpolynucleotides in samples. Such methods can involve substantially thesame steps as described above for presence/absence detection, e.g.,contacting with probe, hybridizing, and detecting hybridized probe, butusing more quantitative methods and/or comparisons to standards.

[0057] The amount of hybridization between the probe and target can bedetermined by any suitable methods, e.g., PCR, RT-PCR, RACE PCR,Northern blot, polynucleotide microarrays, Rapid-Scan, etc., andincludes both quantitative and qualitative measurements. For furtherdetails, see the hybridization methods described above and below.Determining by such hybridization whether the target is differentiallyexpressed (e.g., up-regulated or down-regulated) in the sample can alsobe accomplished by any effective means. For instance, the target'sexpression pattern in the sample can be compared to its pattern in aknown standard, such as in a normal tissue, or it can be compared toanother gene in the same sample. When a second sample is utilized forthe comparison, it can be a sample of normal tissue that is known not tocontain diseased cells. The comparison can be performed on samples whichcontain the same amount of RNA (such as polyadenylated RNA or totalRNA), or, on RNA extracted from the same amounts of starting tissue.Such a second sample can also be referred to as a control or standard.Hybridization can also be compared to a second target in the same tissuesample. Experiments can be performed that determine a ratio between thetarget nucleic acid and a second nucleic acid (a standard or control),e.g., in a normal tissue. When the ratio between the target and controlare substantially the same in a normal and sample, the sample isdetermined or diagnosed not to contain cells. However, if the ratio isdifferent between the normal and sample tissues, the sample isdetermined to contain cancer cells. The approaches can be combined, andone or more second samples, or second targets can be used. Any secondtarget nucleic acid can be used as a comparison, including“housekeeping” genes, such as beta-actin, alcohol dehydrogenase, or anyother gene whose expression does not vary depending upon the diseasestatus of the cell.

[0058] Methods of Identifying Polymorphisms, Mutations, etc., ofTbx3-pr408

[0059] Polynucleotides of the present invention can also be utilized toidentify mutant alleles, SNPs, gene rearrangements and modifications,and other polymorphisms of the wild-type gene. Mutant alleles,polymorphisms, SNPs, etc., can be identified and isolated from cancersthat are known, or suspected to have, a genetic component.Identification of such genes can be carried out routinely (see, abovefor more guidance), e.g., using PCR, hybridization techniques, directsequencing, mismatch reactions (see, e.g., above), RFLP analysis, SSCP(e.g., Orita et al., Proc. Natl. Acad. Sci., 86:2766, 1992), etc., wherea polynucleotide having a sequence selected from SEQ ID NO 1 is used asa probe. The selected mutant alleles, SNPs, polymorphisms, etc., can beused diagnostically to determine whether a subject has, or issusceptible to a disorder associated with Tbx3-pr408, as well as todesign therapies and predict the outcome of the disorder. Methodsinvolve, e.g., diagnosing a disorder associated with Tbx3-pr408 ordetermining susceptibility to a disorder, comprising, detecting thepresence of a mutation in a gene represented by a polynucleotideselected from SEQ ID NO 1. The detecting can be carried out by anyeffective method, e.g., obtaining cells from a subject, determining thegene sequence or structure of a target gene (using, e.g., mRNA, cDNA,genomic DNA, etc), comparing the sequence or structure of the targetgene to the structure of the normal gene, whereby a difference insequence or structure indicates a mutation in the gene in the subject.Polynucleotides can also be used to test for mutations, SNPs,polymorphisms, etc., e.g., using mismatch DNA repair technology asdescribed in U.S. Pat. No. 5,683,877; U.S. Pat. No. 5,656,430; Wu etal., Proc. Natl. Acad. Sci., 89:8779-8783, 1992.

[0060] The present invention also relates to methods of detectingpolymorphisms in Tbx3-pr408, comprising, e.g., comparing the structureof: genomic DNA comprising all or part of Tbx3-pr408, mRNA comprisingall or part of Tbx3-pr408, cDNA comprising all or part of Tbx3-pr408, ora polypeptide comprising all or part of Tbx3-pr408, with the structureof Tbx3-pr408 gene. The methods can be carried out on a sample from anysource, e.g., cells, tissues, body fluids, blood, urine, stool, hair,egg, sperm, cerebral spinal fluid, etc.

[0061] These methods can be implemented in many different ways. Forexample, “comparing the structure” steps include, but are not limitedto, comparing restriction maps, nucleotide sequences, amino acidsequences, RFLPs, Dnase sites, DNA methylation fingerprints (e.g., U.S.Pat. No. 6,214,556), protein cleavage sites, molecular weights,electrophoretic mobilities, charges, ion mobility, etc., between astandard Tbx3-pr408 and a test Tbx3-pr408. The term “structure” canrefer to any physical characteristics or configurations which can beused to distinguish between nucleic acids and polypeptides. The methodsand instruments used to accomplish the comparing step depends upon thephysical characteristics which are to be compared. Thus, varioustechniques are contemplated, including, e.g., sequencing machines (bothamino acid and polynucleotide), electrophoresis, mass spectrometer (U.S.Pat. Nos. 6,093,541, 6,002,127), liquid chromatography, HPLC, etc.

[0062] To carry out such methods, “all or part” of the gene orpolypeptide can be compared. For example, if nucleotide sequencing isutilized, the entire gene can be sequenced, including promoter, introns,and exons, or only parts of it can be sequenced and compared, e.g., exon1, exon 2, etc.

[0063] Mutagenesis

[0064] Mutated polynucleotide sequences of the present invention areuseful for various purposes, e.g., to create mutations of thepolypeptides they encode, to identify functional regions of genomic DNA,to produce probes for screening libraries, etc. Mutagenesis can becarried out routinely according to any effective method, e.g.,oligonucleotide-directed (Smith, M., Ann. Rev. Genet. 19:423-463, 1985),degenerate oligonucleotide-directed (Hill et al., Method Enzymology,155:558-568, 1987), region-specific (Myers et al., Science, 229:242-246,1985; Derbyshire et al., Gene, 46:145, 1986; Ner et al., DNA, 7:127,1988), linker-scanning (McKnight and Kingsbury, Science, 217:316-324,1982), directed using PCR, recursive ensemble mutagenesis (Arkin andYourvan, Proc. Natl. Acad. Sci., 89:7811-7815, 1992), random mutagenesis(e.g., U.S. Pat. Nos. 5,096,815; 5,198,346; and 5,223,409),site-directed mutagenesis (e.g., Walder et al., Gene, 42:133, 1986;Bauer et al., Gene, 37:73, 1985; Craik, Bio Techniques, January 1985,12-19; Smith et al., Genetic Engineering: Principles and Methods, PlenumPress, 1981), phage display (e.g., Lowman et al., Biochem.30:10832-10837, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPOPublication WO 92/06204), etc. Desired sequences can also be produced bythe assembly of target sequences using mutually priming oligonucleotides(Uhlmann, Gene, 71:29-40, 1988). For directed mutagenesis methods,analysis of the three-dimensional structure of the Tbx3-pr408polypeptide can be used to guide and facilitate making mutants whicheffect polypeptide activity. Sites of substrate-enzyme interaction orother biological activities can also be determined by analysis ofcrystal structure as determined by such techniques as nuclear magneticresonance, crystallography or photoaffinity labeling. See, for example,de Vos et al., Science 255:306-312, 1992; Smith et al., J. Mol. Biol.224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992.

[0065]FIG. 1 provides guidance on mutations that can be made inTbx3-pr408, e.g., changing a non-conserved amino acid, e., at amino acidposition 295 and/or 609.

[0066] In addition, libraries of Tbx3-pr408 and fragments thereof can beused for screening and selection of Tbx3-pr408 variants. For instance, alibrary of coding sequences can be generated by treating adouble-stranded DNA with a nuclease under conditions where the nickingoccurs, e.g., only once per molecule, denaturing the double-strandedDNA, renaturing it to for double-stranded DNA that can includesense/antisense pairs from different nicked products, removingsingle-stranded portions from reformed duplexes by treatment with S1nuclease, and ligating the resulting DNAs into an expression vecore. Bythis method, xpression libraries can be made comprising “mutagenized”Tbx3-pr408. The entire coding sequence or parts thereof can be used.

[0067] Polynucleotide Expression, Polypeptides Produced Thereby, andSpecific-binding Partners Thereto.

[0068] A polynucleotide according to the present invention can beexpressed in a variety of different systems, in vitro and in vivo,according to the desired purpose. For example, a polynucleotide can beinserted into an expression vector, introduced into a desired host, andcultured under conditions effective to achieve expression of apolypeptide coded for by the polynucleotide, to search for specificbinding partners. Effective conditions include any culture conditionswhich are suitable for achieving production of the polypeptide by thehost cell, including effective temperatures, pH, medium, additives tothe media in which the host cell is cultured (e.g., additives whichamplify or induce expression such as butyrate, or methotrexate if thecoding polynucleotide is adjacent to a dhfr gene), cycloheximide, celldensities, culture dishes, etc. A polynucleotide can be introduced intothe cell by any effective method including, e.g., naked DNA, calciumphosphate precipitation, electroporation, injection, DEAE-Dextranmediated transfection, fusion with liposomes, association with agentswhich enhance its uptake into cells, viral transfection. A cell intowhich a polynucleotide of the present invention has been introduced is atransformed host cell. The polynucleotide can be extrachromosomal orintegrated into a chromosome(s) of the host cell. It can be stable ortransient. An expression vector is selected for its compatibility withthe host cell. Host cells include, mammalian cells, e.g., COS, CV1, BHK,CHO, HeLa, LTK, NIH 3T3, insect cells, such as Sf9 (S. frugipeda) andDrosophila, bacteria, such as E. coli, Streptococcus, bacillus, yeast,such as Sacharomyces, S. cerevisiae, fungal cells, plant cells,embryonic or adult stem cells (e.g., mammalian, such as mouse or human).

[0069] Expression control sequences are similarly selected for hostcompatibility and a desired purpose, e.g., high copy number, highamounts, induction, amplification, controlled expression. Othersequences which can be employed include enhancers such as from SV40,CMV, RSV, inducible promoters, cell-type specific elements, or sequenceswhich allow selective or specific cell expression. Promoters that can beused to drive its expression, include, e.g., the endogenous promoter,MMTV, SV40, trp, lac, tac, or T7 promoters for bacterial hosts; or alphafactor, alcohol oxidase, or PGH promoters for yeast. RNA promoters canbe used to produced RNA transcripts, such as T7 or SP6. See, e.g.,Melton et al., Polynucleotide Res., 12(18):7035-7056, 1984; Dunn andStudier. J. Mol. Bio., 166:477-435, 1984; U.S. Pat. No. 5,891,636;Studier et al., Gene Expression Technology, Methods in Enzymology,85:60-89, 1987. In addition, as discussed above, translational signals(including in-frame insertions) can be included.

[0070] When a polynucleotide is expressed as a heterologous gene in atransfected cell line, the gene is introduced into a cell as describedabove, under effective conditions in which the gene is expressed. Theterm “heterologous” means that the gene has been introduced into thecell line by the “hand-of-man.” Introduction of a gene into a cell lineis discussed above. The transfected (or transformed) cell expressing thegene can be lysed or the cell line can be used intact.

[0071] For expression and other purposes, a polynucleotide can containcodons found in a naturally-occurring gene, transcript, or cDNA, forexample, e.g., as set forth in SEQ ID NO 1, or it can contain degeneratecodons coding for the same amino acid sequences. For instance, it may bedesirable to change the codons in the sequence to optimize the sequencefor expression in a desired host. See, e.g., U.S. Pat. Nos. 5,567,600and 5,567,862.

[0072] A polypeptide according to the present invention can be recoveredfrom natural sources, transformed host cells (culture medium or cells)according to the usual methods, including, detergent extraction (e.g.,non-ionic detergent, Triton X-100, CHAPS, octylglucoside, IgepalCA-630), ammonium sulfate or ethanol precipitation, acid extraction,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, hydroxyapatitechromatography, lectin chromatography, gel electrophoresis. Proteinrefolding steps can be used, as necessary, in completing theconfiguration of the mature protein. Finally, high performance liquidchromatography (HPLC) can be employed for purification steps. Anotherapproach is express the polypeptide recombinantly with an affinity tag(Flag epitope, HA epitope, myc epitope, 6×His, maltose binding protein,chitinase, etc) and then purify by anti-tag antibody-conjugated affinitychromatography.

[0073] The present invention also relates to antibodies, and otherspecific-binding partners, which are specific for polypeptides encodedby polynucleotides of the present invention, e.g., Tbx3-pr408.Antibodies, e.g., polyclonal, monoclonal, recombinant, chimeric,humanized, single-chain, Fab, and fragments thereof, can be preparedaccording to any desired method. See, also, screening recombinantimmunoglobulin libraries (e.g., Orlandi et al., Proc. Natl. Acad. Sci.,86:3833-3837, 1989; Huse et al., Science, 256:1275-1281, 1989); in vitrostimulation of lymphocyte populations; Winter and Milstein, Nature, 349:293-299, 1991. The antibodies can be IgM, IgG, subtypes, IgG2a, IgG1,etc. Antibodies, and immune responses, can also be generated byadministering naked DNA See, e.g., U.S. Pat. Nos. 5,703,055; 5,589,466;5,580,859. Antibodies can be used from any source, including, goat,rabbit, mouse, chicken (e.g., IgY; see, Duan, W0/029444 for methods ofmaking antibodies in avian hosts, and harvesting the antibodies from theeggs). An antibody specific for a polypeptide means that the antibodyrecognizes a defined sequence of amino acids within or including thepolypeptide. Other specific binding partners include, e.g., aptamers andPNA. antibodies can be prepared against specific epitopes or domains ofTbx3-pr408, e.g., amino acids 596-608 of SEQ ID NO 2, or peptidescomprising amino acids 220-221 (e.g., where the Tbx3-pr408 transcript isto be distinguished from the Tbx3 transcripts which contain the 20-aminacid insertion).

[0074] The preparation of polyclonal antibodies is well-known to thoseskilled in the art. See, for example, Green et al., Production ofPolyclonal Antisera, in IMMUNOCHEMICAL PROTOCOLS (Manson, ed.), pages1-5 (Humana Press 1992); Coligan et al., Production of PolyclonalAntisera in Rabbits, Rats, Mice and Hamsters, in CURRENT PROTOCOLS INIMMUNOLOGY, section 2.4.1 (1992). The preparation of monoclonalantibodies likewise is conventional. See, for example, Kohler &Milstein, Nature 256:495 (1975); Coligan et al., sections 2.5.1-2.6.7;and Harlow et al., ANTIBODIES: A LABORATORY MANUAL, page 726 (ColdSpring Harbor Pub. 1988).

[0075] Antibodies can also be humanized, e.g., where they are to be usedtherapeutically. Humanized monoclonal antibodies are produced bytransferring mouse complementarity determining regions from heavy andlight variable chains of the mouse immunoglobulin into a human variabledomain, and then substituting human residues in the framework regions ofthe murine counterparts. The use of antibody components derived fromhumanized monoclonal antibodies obviates potential problems associatedwith the immunogenicity of murine constant regions. General techniquesfor cloning murine immunoglobulin variable domains are described, forexample, by Orlandi et al., Proc. Nat'l Acad. Sci. USA 86:3833 (1989),which is hereby incorporated in its entirety by reference. Techniquesfor producing humanized monoclonal antibodies are described, forexample, in U.S. Pat. No. 6,054,297, Jones et al., Nature 321: 522(1986); Riechmann et al., Nature 332: 323 (1988); Verhoeyen et al.,Science 239: 1534 (1988); Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285 (1992); Sandhu, Crit. Rev. Biotech. 12: 437 (1992); and Singer etal., J. lmnmunol. 150: 2844 (1993).

[0076] Antibodies of the invention also may be derived from humanantibody fragments isolated from a combinatorial immunoglobulin library.See, for example, Barbas et al., METHODS: A COMPANION TO METHODS INENZYMOLOGY, VOL. 2, page 119 (1991); Winter et al., Ann. Rev. Immunol.12: 433 (1994). Cloning and expression vectors that are useful forproducing a human immunoglobulin phage library can be obtainedcommercially, for example, from STRATAGENE Cloning Systems (La Jolla,Calif.).

[0077] In addition, antibodies of the present invention may be derivedfrom a human monoclonal antibody. Such antibodies are obtained fromtransgenic mice that have been “engineered” to produce specific humanantibodies in response to antigenic challenge. In this technique,elements of the human heavy and light chain loci are introduced intostrains of mice derived from embryonic stem cell lines that containtargeted disruptions of the endogenous heavy and light chain loci. Thetransgenic mice can synthesize human antibodies specific for humanantigens and can be used to produce human antibody-secreting hybridomas.Methods for obtaining human antibodies from transgenic mice aredescribed, e.g., in Green et al., Nature Genet. 7:13 (1994); Lonberg etal., Nature 368:856 (1994); and Taylor et al., Int. Immunol. 6:579(1994).

[0078] Antibody fragments of the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ofnucleic acid encoding the fragment. Antibody fragments can be obtainedby pepsin or papain digestion of whole antibodies by conventionalmethods. For example, antibody fragments can be produced by enzymaticcleavage of antibodies with pepsin to provide a 5S fragment denotedF(ab′).sub.2. This fragment can be further cleaved using a thiolreducing agent, and optionally a blocking group for the sulfhydrylgroups resulting from cleavage of disulfide linkages, to produce 3.5SFab′ monovalent fragments. Alternatively, an enzymatic cleavage usingpepsin produces two monovalent Fab′ fragments and an Fc fragmentdirectly. These methods are described, for example, by Goldenberg, U.S.Pat. No. 4,036,945 and No. 4,331,647, and references contained therein.These patents are hereby incorporated in their entireties by reference.See also Nisoiihoff et al., Arch. Biochem. Biophys. 89:230 (1960);Porter, Biochem. J. 73:119 (1959); Edelman etal, METHODS IN ENZYMOLOGY,VOL. 1, page 422 (Academic Press 1967); and Coligan et al. at sections2.8.1-2.8.10 and 2.10.1-2.10.4.

[0079] Other methods of cleaving antibodies, such as separation of heavychains to form monovalent light-heavy chain fragments, further cleavageof fragments, or other enzymatic, chemical, or genetic techniques canalso be used. For example, Fv fragments comprise an association ofV.sub.H and V.sub.L chains. This association may be noncovalent, asdescribed in Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659 (1972).Alternatively, the variable chains can be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde. See,e.g., Sandhu, supra. Preferably, the Fv fragments comprise V.sub.H andV.sub.L chains connected by a peptide linker. These single-chain antigenbinding proteins (sFv) are prepared by constructing a structural genecomprising nucleic acid sequences encoding the V.sub.H and V.sub.Ldomains connected by an oligonucleotide. The structural gene is insertedinto an expression vector, which is subsequently introduced into a hostcell such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by Whitlow etal., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 97(1991); Bird etal.,Science 242:423-426 (1988); Ladneret al., U.S. Pat.No. 4,946,778; Pack et al., Bio/Technology 11: 1271-77 (1993); andSandhu, supra.

[0080] Another form of an antibody fragment is a peptide coding for asingle complementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick et al.,METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 106 (1991).

[0081] The term “antibody” as used herein includes intact molecules aswell as fragments thereof, such as Fab, F(ab′)2, and Fv which arecapable of binding to an epitopic determinant present in Bin1polypeptide. Such antibody fragments retain some ability to selectivelybind with its antigen or receptor. The term “epitope” refers to anantigenic determinant on an antigen to which the paratope of an antibodybinds. Epitopic determinants usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Antibodies can be preparedagainst specific epitopes or polypeptide domains.

[0082] Antibodies which bind to Tbx3-pr408 polypeptides of the presentinvention can be prepared using an intact polypeptide or fragmentscontaining small peptides of interest as the immunizing antigen. Forexample, it may be desirable to produce antibodies that specificallybind to the N- or C-terminal domains of Tbx3-pr408. The polypeptide orpeptide used to immunize an animal which is derived from translated cDNAor chemically synthesized which can be conjugated to a carrier protein,if desired. Such commonly used carriers which are chemically coupled tothe immunizing peptide include keyhole limpet hemocyanin (KLH),thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.

[0083] Polyclonal or monoclonal antibodies can be further purified, forexample, by binding to and elution from a matrix to which thepolypeptide or a peptide to which the antibodies were raised is bound.Those of skill in the art will know of various techniques common in the.immunology arts for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies (See for example, Coligan,et al., Unit 9, Current Protocols in Immunology, Wiley Interscience,1994, incorporated by reference).

[0084] Anti-idiotype technology can also be used to produce inventionmonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody.

[0085] Methods of Detecting Polypeptides

[0086] Polypeptides coded for by Tbx3-pr408 of the present invention canbe detected, visualized, determined, quantitated, etc. according to anyeffective method. useful methods include, e.g., but are not limited to,immunoassays, RIA (radioimmunoassay), ELISA,(enzyme-linked-immunosorbent assay), immunoflourescence, flow cytometry,histology, electron microscopy, light microscopy, in situ assays,immunoprecipitation, Western blot, etc.

[0087] Immunoassays may be carried in liquid or on biological support.For instance, a sample (e.g., blood, stool, urine, cells, tissue,cerebral spinal fluid, body fluids, etc.) can be brought in contact withand immobilized onto a solid phase support or carrier such asnitrocellulose, or other solid support that is capable of immobilizingcells, cell particles or soluble proteins. The support may then bewashed with suitable buffers followed by treatment with the detectablylabeled Tbx3-pr408 specific antibody. The solid phase support can thenbe washed with a buffer a second time to remove unbound antibody. Theamount of bound label on solid support may then be detected byconventional means.

[0088] A “solid phase support or carrier” includes any support capableof binding an antigen, antibody, or other specific binding partner.Supports or carriers include glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, and magnetite. A support material can have anystructural or physical configuration. Thus, the support configurationmay be spherical, as in a bead, or cylindrical, as in the inside surfaceof a test tube, or the external surface of a rod. Alternatively, thesurface may be flat such as a sheet, test strip, etc. Preferred supportsinclude polystyrene beads

[0089] One of the many ways in which gene peptide-specific antibody canbe detectably labeled is by linking it to an enzyme and using it in anenzyme immunoassay (EIA). See, e.g., Voller, A., “The Enzyme LinkedImmunosorbent Assay (ELISA),” 1978, Diagnostic Horizons 2, 1-7,Microbiological Associates Quarterly Publication, Walkersville, Md.);Voller, A. et al., 1978, J. Clin. Pathol. 31, 507-520; Butler, J. E.,1981, Meth. Enzymol. 73, 482-523; Maggio, E. (ed.), 1980, EnzymeImmunoassay, CRC Press, Boca Raton, Fla. The enzyme which is bound tothe antibody will react with an appropriate substrate, preferably achromogenic substrate, in such a manner as to produce a chemical moietythat can be detected, for example, by spectrophotometric, fluorimetricor by visual means. Enzymes that can be used to detectably label theantibody include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, .alpha.-glycerophosphate, dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, .beta.-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by calorimetricmethods that employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

[0090] Detection may also be accomplished using any of a variety ofother immunoassays. For example, by radioactively labeling theantibodies or antibody fragments, it is possible to detect Tbx3-pr408peptides through the use of a radioimmunoassay (RIA). See, e.g.,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986. Theradioactive isotope can be detected by such means as the use of a gammacounter or a scintillation counter or by autoradiography.

[0091] It is also possible to label the antibody with a fluorescentcompound. When the fluorescently labeled antibody is exposed to light ofthe proper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. Theantibody can also be detectably labeled using fluorescence emittingmetals such as those in the lanthanide series. These metals can beattached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

[0092] The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples of usefulchemiluminescent labeling compounds are luminol, isoluminol, theromaticacridinium ester, imidazole, acridinium salt and oxalate ester.

[0093] Likewise, a bioluminescent compound may be used to label theantibody of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase and aequorin.

[0094] Diagnostic

[0095] The present invention also relates to methods and compositionsfor diagnosing a developmental disorder, or determining susceptibilityto a disorder, using polynucleotides, polypeptides, and specific-bindingpartners of the present invention to detect, assess, determine, etc.,Tbx3-pr408. In such methods, the gene can serve as a marker for thedisorder, e.g., where the gene, when mutant, is a direct cause of thedisorder; where the gene is affected by another gene(s) which isdirectly responsible for the disorder, e.g., when the gene is part ofthe same signaling pathway as the directly responsible gene; and, wherethe gene is chromosomally linked to the gene(s) directly responsible forthe disorder, and segregates with it. Many other situations arepossible. To detect, assess, determine, etc., a probe specific for thegene can be employed as described above and below. Any method ofdetecting and/or assessing the gene can be used, including detectingexpression of the gene using polynucleotides, antibodies, or otherspecific-binding partners.

[0096] The present invention relates to methods of diagnosing a disorderassociated with Tbx3-pr408 (e.g., of the prostate, heart, muscle, lung,or adrenal gland), or determining a subject's susceptibility to suchdisorder, comprising, e.g., assessing the expression of Tbx3-pr408 in atissue sample comprising tissue or cells suspected of having thedisorder.

[0097] The phrase “diagnosing” indicates that it is determined whetherthe sample has the disorder. A “disorder” means, e.g., any abnormalcondition as in a disease or malady. “Determining a subject'ssusceptibility to a disease or disorder” indicates that the subject isassessed for whether s/he is predisposed to get such a disease ordisorder, where the predisposition is indicated by abnormal expressionof the gene (e.g., gene mutation, gene expression pattern is not normal,etc.). Predisposition or susceptibility to a disease may result when asuch disease is influenced by epigenetic, environmental, etc., factors.This includes prenatal screening where samples from the fetus or embryo(e.g., via amniocentesis or CV sampling) are analyzed for the expressionof the gene.

[0098] By the phrase “assessing expression of Tbx3-pr408,” it is meantthat the functional status of the gene is evaluated. This includes, butis not limited to, measuring expression levels of said gene, determiningthe genomic structure of said gene, determining the mRNA structure oftranscripts from said gene, or measuring the expression levels ofpolypeptide coded for by said gene. Thus, the term “assessingexpression” includes evaluating the all aspects of the transcriptionaland translational machinery of the gene. For instance, if a promoterdefect causes, or is suspected of causing, the disorder, then a samplecan be evaluated (i.e., “assessed”) by looking (e.g., sequencing orrestriction mapping) at the promoter sequence in the gene, by detectingtranscription products (e.g., RNA), by detecting translation product(e.g., polypeptide). Any measure of whether the gene is functional canbe used, including, polypeptide, polynucleotide, and functional assaysfor the gene's biological activity.

[0099] In making the assessment, it can be useful to compare the resultsto a normal gene, e.g., a gene which is not associated with thedisorder. The nature of the comparison can be determined routinely,depending upon how the assessing is accomplished. If, for example, themRNA levels of a sample is detected, then the mRNA levels of a normalcan serve as a comparison, or a gene which is known not to be affectedby the disorder. Methods of detecting mRNA are well known, and discussedabove, e.g., but not limited to, Northern blot analysis, polymerasechain reaction (PCR), reverse transcriptase PCR, RACE PCR, etc.Similarly, if polypeptide production is used to evaluate the gene, thenthe polypeptide in a normal tissue sample can be used as a comparison,or, polypeptide from a different gene whose expression is known not tobe affected by the disorder. These are only examples of how such amethod could be carried out.

[0100] Assessing the effects of therapeutic and preventativeinterventions (e.g., administration of a drug, chemotherapy, radiation,etc.) on disorders is a major effort in drug discovery, clinicalmedicine, and pharmacogenomics. The evaluation of therapeutic andpreventative measures, whether experimental or already in clinical use,has broad applicability, e.g., in clinical trials, for monitoring thestatus of a patient, for analyzing and assessing animal models, and inany scenario involving cancer treatment and prevention. Analyzing theexpression profiles of polynucleotides of the present invention can beutilized as a parameter by which interventions are judged and measured.Treatment of a disorder can change the expression profile in some mannerwhich is prognostic or indicative of the drug's effect on it. Changes inthe profile can indicate, e.g., drug toxicity, return to a normal level,etc. Accordingly, the present invention also relates to methods ofmonitoring or assessing a therapeutic or preventative measure (e.g.,chemotherapy, radiation, anti-neoplastic drugs, antibodies, etc.) in asubject having a disorder, or, susceptible to such a disorder,comprising, e.g., detecting the expression levels of Tbx3-pr408. Asubject can be a cell-based assay system, non-human animal model, humanpatient, etc. Detecting can be accomplished as described for the methodsabove and below. By “therapeutic or preventative intervention,” it ismeant, e.g., a drug administered to a patient, surgery, radiation,chemotherapy, and other measures taken to prevent, treat, or diagnose adisorder.

[0101] The present invention also relates to methods of detecting Tbxtranscripts in a tissue sample, comprising, e.g., assessing theexpression of a Tbx3-pr408 transcript in a tissue sample, and assessingthe expression of other transcripts of the Tbx3 gene, such as thoserepresented by NM_(—)005996 (SEQ ID NO 3), XM_(—)016321 (SEQ ID NO 4),and NM_(—)016569 (SEQ ID NO 5). Tbx3 transcripts which contain about a20 amino acid insertion at about amino acid 220 are referred to “Tbx3transcripts comprising a T box insertion.” Examples are NM_(—)005996(SEQ ID NO 3) and XM_(—)016321 (SEQ ID NO 4). Assessing can beaccomplished as described above. This method can be used to determinethe relative levels of Tbx3-pr408 as compared to other Tbx3 transcripts.For instance, as described above, Tbx3-pr408 is more abundant in mosttissues than NM_(—)005996 or XM_(—)016321. Patients having mammary-ulnarsyndrome can be assessed for the relative levels of both transcripttypes. If PCR is used to determine mRNA expression, the followingprimers can be used which span the insertion, e.g., forward primerhaving the sequence of about 1023-1048 (SEQ ID NO 1), and a reverseprimer which is complementary to the sequence of about 1230-1255. Ifboth transcripts are present in the sample, two different bands will bedetected, the larger corresponding to NM_(—)005996 or XM_(—)016321, andthe smaller to Tbx3-pr408.

[0102] Identifying Agent Methods

[0103] The present invention relates to compositions and methods ofmodulating Tbx3-pr48 gene or polypeptide. Assays can be performed onwhole cells, lysates, isolated Tbx3-pr408, etc.

[0104] For example, the present invention also relates to methods ofidentifying agents that modulate the expression of Tbx3-pr408 in a cellpopulation, comprising, in any effective order, one or more of thefollowing steps, e.g., contacting a cell population with a test agentunder conditions effective for said test agent to modulate theexpression of Tbx3-pr408 in said cell population, and determiningwhether said test agent modulates said Tbx3-pr408. An agent can modulateexpression of Tbx3-pr408 at any level, including transcription,translation, and/or perdurance of the nucleic acid or polypeptide (e.g.,degradation, stability, etc.) product in the cell.

[0105] In addition, the present invention also relates to methods ofidentifying agents that modulate the biological activity of Tbx3-pr408polypeptide in a cell population, lysate, etc., comprising, in anyeffective order, one or more of the following steps, e.g., contactingsaid polypeptide with a test agent under conditions effective for saidtest agent to modulate the biological activity of said polypeptide, anddetermining whether said test agent modulates said biological activity.

[0106] Contacting the cell population with the test agent can beaccomplished by any suitable method and/or means that places the agentin a position to functionally control expression or biological activityof Tbx3-pr408 present in cells within the population, or in the samplemixture. Functional control indicates that the agent can exert itsphysiological effect on the cell through whatever mechanism it works.The choice of the method and/or means can depend upon the nature of theagent and the condition and type of the cell population (such as, invivo, in vitro, organ explants, etc.). For instance, if the cellpopulation is an in vitro cell culture, the agent can be contacted withthe cells by adding it directly into the culture medium. If the agentcannot dissolve readily in an aqueous medium, it can be incorporatedinto liposomes, or another lipophilic carrier, and then administered tothe cell culture. Contact can also be facilitated by incorporation ofagent with carriers and delivery molecules and complexes, by injection,by infusion, etc.

[0107] After the agent has been administered in such a way that it cangain access to the cells or polypeptide, it can be determined whetherthe test agent modulates Tbx3-pr408 expression or biological activity.Modulation can be of any type, quality, or quantity, e.g., increase,facilitate, enhance, up-regulate, stimulate, activate, amplify, augment,induce, decrease, down-regulate, diminish, lessen, reduce, etc. Themodulatory quantity can also encompass any value, e.g., 1%, 5%, 10%,50%, 75%, 1-fold, 2-fold, 5-fold, 10-fold, 100-fold, etc. To modulateTbx3-pr408 expression means, e.g., that the test agent has an effect onits expression, e.g., to effect the amount of transcription, to effectRNA splicing, to effect translation of the RNA into polypeptide, toeffect RNA or polypeptide stability, to effect polyadenylation or otherprocessing of the RNA, to effect post-transcriptional orpost-translational processing, etc. To modulate biological activitymeans, e.g., that the activity of the polypeptide is changed incomparison to its normal activity in the absence of the agent. Thiseffect includes, increase, decrease, block, inhibit, enhance, etc.Biological activities of Tbx3-pr408 included, e.g., transcriptionregulatory activity, DNA-binding activity, etc. Assays for measuringthese activities were discussed previously.

[0108] A test agent can be of any molecular composition, e.g., chemicalcompounds, biomolecules, such as polypeptides, lipids, nucleic acids(e.g., antisense to a polynucleotide sequence selected from SEQ ID NO1), carbohydrates, antibodies, ribozymes, double-stranded RNA, etc. Forexample, if a polypeptide to be modulated is a cell-surface molecule, atest agent can be an antibody that specifically recognizes it and, e.g.,causes the polypeptide to be internalized, leading to its downregulation on the surface of the cell. Such an effect does not have tobe permanent, but can require the presence of the antibody to continuethe down-regulatory effect. Antisense Tbx3-pr408 can also be used astest agents to modulate gene expression.

[0109] The entire Tbx3-pr408 polypeptide can be used, orbiologically-active fragments thereof. If transcriptional activity isbeing measured, then a biologically-active fragment would be one thatretains transcriptional regulatory activity, and so on. In some cases,biologically-active fragments can comprise Tbx3-pr408 specific sequence.By this, it is meant that fragments common to other Tbx3 transcripts(e.g., NM_(—)005996, XM_(—)016321, and NM_(—)016569) are excluded. Anexample of such a fragment would comprise amino acids from about596-608, e.g., 1-650, 50-650, 50-723, 300-650 (e.g., fragments deletingthe DNA-binding domain), 300-723, etc.

[0110] Therapeutics

[0111] Selective polynucleotides, polypeptides, and specific-bindingpartners thereto, can be utilized in therapeutic applications,especially to treat diseases and conditions of associated withTbx-pr408, e.g., mammary-ulnar disease. Useful methods include, but arenot limited to, immunotherapy (e.g., using specific-binding partners topolypeptides), vaccination (e.g., using a selective polypeptide or anaked DNA encoding such polypeptide), protein or polypeptide replacementtherapy, gene therapy (e.g., germ-line correction, antisense), etc.

[0112] Various immunotherapeutic approaches can be used. For instance,unlabeled antibody that specifically recognizes a tissue-specificantigen can be used to stimulate the body to destroy or attack thecancer, to cause down-regulation, to produce complement-mediated lysis,to inhibit cell growth, etc., of target cells which display the antigen,e.g., analogously to how c-erbB-2 antibodies are used to treat breastcancer. In addition, antibody can be labeled or conjugated to enhanceits deleterious effect, e.g., with radionuclides and other energyemitting entitities, toxins, such as ricin, exotoxin A (ETA), anddiphtheria, cytotoxic or cytostatic agents, immunomodulators,chemotherapeutic agents, etc. See, e.g., U.S. Pat. No. 6,107,090.

[0113] An antibody or other specific-binding partner can be conjugatedto a second molecule, such as a cytotoxic agent, and used for targetingthe second molecule to a tissue-antigen positive cell (Vitetta, E. S. etal., 1993, Immunotoxin therapy, in DeVita, Jr., V. T. et al., eds,Cancer: Principles and Practice of Oncology, 4th ed., J. B. LippincottCo., Philadelphia, 2624-2636). Examples of cytotoxic agents include, butare not limited to, antimetabolites, alkylating agents, anthracyclines,antibiotics, anti-mitotic agents, radioisotopes and chemotherapeuticagents. Further examples of cytotoxic agents include, but are notlimited to ricin, doxorubicin, daunorubicin, taxol, ethidium bromide,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine,dihydroxy anthracin dione, actinomycin D, 1-dehydrotestosterone,diptheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, elongationfactor-2 and glucocorticoid. Techniques for conjugating therapeuticagents to antibodies are well.

[0114] In addition to immunotherapy, polynucleotides and polypeptidescan be used as targets for non-immunotherapeutic applications, e.g.,using compounds which interfere with function, expression (e.g.,antisense as a therapeutic agent), assembly, etc. RNA interference canbe used in vivtro and in vivo to silence Tbx3-pr408 when its expressioncontributes to a disease (but also for other purposes, e.g., to identifythe gene's function to change a developmental pathway of a cell, etc.).See, e.g., Sharp and Zamore, Science, 287:2431-2433, 2001; Grishok etal., Science, 287:2494, 2001.

[0115] Delivery of therapeutic agents can be achieved according to anyeffective method, including, liposomes, viruses, plasmid vectors,bacterial delivery systems, orally, systemically, etc. Therapeuticagents of the present invention can be administered in any form by anyeffective route, including, e.g., oral, parenteral, enteral,intraperitoneal, topical, transdermal (e.g., using any standard patch),ophthalmic, nasally, local, non-oral, such as aerosal, inhalation,subcutaneous, intramuscular, buccal, sublingual, rectal, vaginal,intra-arterial, and intrathecal, etc. They can be administered alone, orin combination with any ingredient(s), active or inactive.

[0116] In addition to therapeutics, per se, the present invention alsorelates to methods of treating a disease showing altered expression ofTbx3-pr408, comprising, e.g., administering to a subject in need thereofa therapeutic agent which is effective for regulating expression of saidTbx3-pr408 and/or which is effective in treating said disease. The term“treating” is used conventionally, e.g., the management or care of asubject for the purpose of combating, alleviating, reducing, relieving,improving the condition of, etc., of a disease or disorder. By thephrase “altered expression,” it is meant that the disease is associatedwith a mutation in the gene, or any modification to the gene (orcorresponding product) which affects its normal function. Thus,expression of Tbx3-pr408 refers to, e.g., transcription, translation,splicing, stability of the mRNA or protein product, activity of the geneproduct, differential expression, etc.

[0117] Any agent which “treats” the disease can be used. Such an agentcan be one which regulates the expression of the Tbx3-pr408. Expressionrefers to the same acts already mentioned, e.g. transcription,translation, splicing, stability of the mRNA or protein product,activity of the gene product, differential expression, etc. Forinstance, if the condition was a result of a complete deficiency of thegene product, administration of gene product to a patient would be saidto treat the disease and regulate the gene's expression. Many otherpossible situations are possible, e.g., where the gene is aberrantlyexpressed, and the therapeutic agent regulates the aberrant expressionby restoring its normal expression pattern.

[0118] Antisense

[0119] Antisense polynucleotide (e.g., RNA) can also be prepared from apolynucleotide according to the present invention, preferably ananti-sense to a sequence of SEQ ID NO 1. Antisense polynucleotide can beused in various ways, such as to regulate or modulate expression of thepolypeptides they encode, e.g., inhibit their expression, for in situhybridization, for therapeutic purposes, for making targeted mutations(in vivo, triplex, etc.) etc. For guidance on administering anddesigning anti-sense, see, e.g., U.S. Pat. Nos. 6,200,960, 6,200,807,6,197,584, 6,190,869, 6,190,661, 6,187,587, 6,168,950, 6,153,595,6,150,162, 6,133,246, 6,117,847, 6,096,722, 6,087,343, 6,040,296,6,005,095, 5,998,383, 5,994,230, 5,891,725, 5,885,970, and 5,840,708. Anantisense polynucleotides can be operably linked to an expressioncontrol sequence. A total length of about 35 bp can be used in cellculture with cationic liposomes to facilitate cellular uptake, but forin vivo use, preferably shorter oligonucleotides are administered, e.g.25 nucleotides.

[0120] Antisense polynucleotides can comprise modified,nonnaturally-occurring nucleotides and linkages between the nucleotides(e.g., modification of the phosphate-sugar backbone; methyl phosphonate,phosphorothioate, or phosphorodithioate linkages; and 2′-O-methyl ribosesugar units), e.g., to enhance in vivo or in vitro stability, to confernuclease resistance, to modulate uptake, to modulate cellulardistribution and compartmentalization, etc. Any effective nucleotide ormodification can be used, including those already mentioned, as known inthe art, etc., e.g., disclosed in U.S. Pat. Nos. 6,133,438; 6,127,533;6,124,445; 6,121,437; 5,218,103 (e.g., nucleoside thiophosphoramidites);4,973,679; Sproat et al., “2′-O-Methyloligoribonucleotides: synthesisand applications,” Oligonucleotides and Analogs A Practical Approach,Eckstein (ed.), IRL Press, Oxford, 1991, 49-86; Iribarren et al.,“2′-O-Alkyl Oligoribonucleotides as Antisense Probes,” Proc. Natl. Acad.Sci. USA, 1990, 87, 7747-7751; Cotton et al., “2′-O-methyl, 2′-O-ethyloligoribonucleotides and phosphorothioate oligodeoxyribonucleotides asinhibitors of the in vitro U7 snRNP-dependent mRNA processing event,”Nucl. Acids Res., 1991, 19, 2629-2635.

[0121] Arrays

[0122] The present invention also relates to an ordered array ofpolynucleotide probes and specific-binding partners (e.g., antibodies)for detecting the expression of Tbx3-pr408 in a sample, comprising, oneor more polynucleotide probes or specific binding partners associatedwith a solid support, wherein each probe is specific for Tbx3-pr408, andthe probes comprise a nucleotide sequence of SEQ ID NO 1 which isspecific for said gene, a nucleotide sequence having sequence identityto SEQ ID NO 1 which is specific for said gene or polynucleotide, orcomplements thereto, or a specific-binding partner which is specific forTbx3-pr408.

[0123] The phrase “ordered array” indicates that the probes are arrangedin an identifiable or position-addressable pattern, e.g., such as thearrays disclosed in U.S. Pat. Nos. 6,156,501, 6,077,673, 6,054 ,270,5,723,320, 5,700,637, WO0991971 1, WO00023803. The probes are associatedwith the solid support in any effective way. For instance, the probescan be bound to the solid support, either by polymerizing the probes onthe substrate, or by attaching a probe to the substrate. Association canbe, covalent, electrostatic, noncovalent, hydrophobic, hydrophilic,noncovalent, coordination, adsorbed, absorbed, polar, etc. When fibersor hollow filaments are utilized for the array, the probes can fill thehollow orifice, be absorbed into the solid filament, be attached to thesurface of the orifice, etc. Probes can be of any effective size,sequence identity, composition, etc., as already discussed.

[0124] Transgenic Animals

[0125] The present invention also relates to transgenic animalscomprising Tbx3-pr408 genes. Such genes, as discussed in more detailbelow, include, but are not limited to, functionally-disrupted genes,mutated genes, ectopically or selectively-expressed genes, inducible orregulatable genes, etc. These transgenic animals can be producedaccording to any suitable technique or method, including homologousrecombination, mutagenesis (e.g., ENU, Rathkolb et al., Exp. Physiol.,85(6):635-644, 2000), and the tetracycline-regulated gene expressionsystem (e.g., U.S. Pat. No. 6,242,667). The term “gene” as used hereinincludes any part of a gene, i.e., regulatory sequences, promoters,enhancers, exons, introns, coding sequences, etc. The Tbx3-pr408 nucleicacid present in the construct or transgene can be naturally-occurringwild-type, polymorphic, or mutated.

[0126] Along these lines, polynucleotides of the present invention canbe used to create transgenic animals, e.g. a non-human animal,comprising at least one cell whose genome comprises a functionaldisruption of Tbx3-pr408. By the phrases “functional disruption” or“functionally disrupted,” it is meant that the gene does not express abiologically-active product. It can be substantially deficient in atleast one functional activity coded for by the gene. Expression of apolypeptide can be substantially absent, i.e., essentially undetectableamounts are made. However, polypeptide can also be made, but which isdeficient in activity, e.g., where only an amino-terminal portion of thegene product is produced.

[0127] The transgenic animal can comprise one or more cells. Whensubstantially all its cells contain the engineered gene, it can bereferred to as a transgenic animal “whose genome comprises” theengineered gene. This indicates that the endogenous gene loci of theanimal has been modified and substantially all cells contain suchmodification.

[0128] Functional disruption of the gene can be accomplished in anyeffective way, including, e.g., introduction of a stop codon into anypart of the coding sequence such that the resulting polypeptide isbiologically inactive (e.g., because it lacks a catalytic domain, aligand binding domain, etc.), introduction of a mutation into a promoteror other regulatory sequence that is effective to turn it off, or reducetranscription of the gene, insertion of an exogenous sequence into thegene which inactivates it (e.g., which disrupts the production of abiologically-active polypeptide or which disrupts the promoter or othertranscriptional machinery), deletion of sequences from the Tbx3-pr408gene, etc. Examples of transgenic animals having functionally disruptedgenes are well known, e.g., as described in U.S. Pat. Nos. 6,239,326,6,225,525, 6,207,878, 6,194,633, 6,187,992, 6,180,849, 6,177,610,6,100,445, 6,087,555, 6,080,910, 6,069,297, 6,060,642, 6,028,244,6,013,858, 5,981,830, 5,866,760, 5,859,314, 5,850,004, 5,817,912,5,789,654, 5,777,195, and 5,569,824. A transgenic animal which comprisesthe functional disruption can also be referred to as a “knock-out”animal, since the biological activity of its Tbx3-pr408 genes has been“knocked-out.” Knock-outs can be homozygous or heterozygous.

[0129] For creating functional disrupted genes, and other genemutations, homologous recombination technology is of special interestsince it allows specific regions of the genome to be targeted. Usinghomologous recombination methods, genes can be specifically-inactivated,specific mutations can be introduced, and exogenous sequences can beintroduced at specific sites. These methods are well known in the art,e.g., as described in the patents above. See, also, Robertson, Biol.Reproduc., 44(2):238-245, 1991. Generally, the genetic engineering isperformed in an embryonic stem (ES) cell, or other pluripotent cell line(e.g., adult stem cells, EG cells), and that genetically-modified cell(or nucleus) is used to create a whole organism. Nuclear transfer can beused in combination with homologous recombination technologies.

[0130] For example, the Tbx3-pr408 locus can be disrupted in mouse EScells using a positive-negative selection method (e.g., Mansour et al.,Nature, 336:348-352, 1988). In this method, a targeting vector can beconstructed which comprises a part of the gene to be targeted. Aselectable marker, such as neomycin resistance genes, can be insertedinto a Tbx3-pr408 exon present in the targeting vector, disrupting it.When the vector recombines with the ES cell genome, it disrupts thefunction of the gene. The presence in the cell of the vector can bedetermined by expression of neomycin resistance. See, e.g., U.S. Pat.No. 6,239,326. Cells having at least one functionally disrupted gene canbe used to make chimeric and germine animals, e.g., animals havingsomatic and/or germ cells comprising the engineered gene. Homozygousknock-out animals can be obtained from breeding heterozygous knock-outanimals. See, e.g., U.S. Pat. No. 6,225,525.

[0131] A transgenic animal, or animal cell, lacking one or morefunctional Tbx3-pr408 genes can be useful in a variety of applications,including, as an animal model for mammary-ulnar disease, and any of theutilities mentioned in any issued U.S. Patent on transgenic animals,including, U.S. Pat. Nos. 6,239,326, 6,225,525, 6,207,878, 6,194,633,6,187,992, 6,180,849, 6,177,610, 6,100,445, 6,087,555, 6,080,910,6,069,297, 6,060,642, 6,028,244, 6,013,858, 5,981,830, 5,866,760,5,859,314, 5,850,004, 5,817,912, 5,789,654, 5,777,195, and 5,569,824

[0132] The present invention also relates to non-human, transgenicanimal whose genome comprises recombinant Tbx3-pr408 nucleic acidoperatively linked to an expression control sequence effective toexpress said coding sequence, e.g., in heart, prostate, lung, adrenal,testes, and/or muscle. Such a transgenic animal can also be referred toas a “knock-in” animal since an exogenous gene has been introduced,stably, into its genome.

[0133] A recombinant Tbx3-pr408 nucleic acid refers to a gene which hasbeen introduced into a target host cell and optionally modified, such ascells derived from animals, plants, bacteria, yeast, etc. A recombinantTbx3-pr408 includes completely synthetic nucleic acid sequences,semi-synthetic nucleic acid sequences, sequences derived from naturalsources, and chimeras thereof. “Operable linkage” has the meaning usedthrough the specification, i.e., placed in a functional relationshipwith another nucleic acid. When a gene is operably linked to anexpression control sequence, as explained above, it indicates that thegene (e.g., coding sequence) is joined to the expression controlsequence (e.g., promoter) in such a way that facilitates transcriptionand translation of the coding sequence. As described above, the phrase“genome” indicates that the genome of the cell has been modified. Inthis case, the recombinant Tbx3-pr408 has been stably integrated intothe genome of the animal. The Tbx3-pr408 nucleic acid in operablelinkage with the expression control sequence can also be referred to asa construct or transgene.

[0134] Any expression control sequence can be used depending on thepurpose. For instance, if selective expression is desired, thenexpression control sequences which limit its expression can be selected.These include, e.g., tissue or cell-specific promoters, introns,enhancers, etc. For various methods of cell and tissue-specificexpression, see, e.g., U.S. Pat. Nos. 6,215,040, 6,210,736, and6,153,427. These also include the endogenous promoter, i.e., the codingsequence can be operably linked to its own promoter. Inducible andregulatable promoters can also be utilized.

[0135] The present invention also relates to a transgenic animal whichcontains a functionally disrupted and a transgene stably integrated intothe animals genome. Such an animal can be constructed using combinationsany of the above- and below-mentioned methods. Such animals have any ofthe aforementioned uses, including permitting the knock-out of thenormal gene and its replacement with a mutated gene. Such a transgenecan be integrated at the endogenous gene locus so that the functionaldisruption and “knock-in” are carried out in the same step.

[0136] In addition to the methods mentioned above, transgenic animalscan be prepared according to known methods, including, e.g., bypronuclear injection of recombinant genes into pronuclei of 1-cellembryos, incorporating an artificial yeast chromosome into embryonicstem cells, gene targeting methods, embryonic stem cell methodology,cloning methods, nuclear transfer methods. See, also, e.g., U.S. Pat.Nos. 4,736,866; 4,873,191; 4,873,316; 5,082,779; 5,304,489; 5,174,986;5,175,384; 5,175,385; 5,221,778; Gordon et al., Proc. Natl. Acad. Sci.,77:7380-7384, 1980; Palmiter et al., Cell, 41:343-345, 1985; Palmiter etal., Ann. Rev. Genet., 20:465-499, 1986; Askew et al., Mol. Cell. Bio.,13:4115-4124, 1993; Games et al. Nature, 373:523-527, 1995; Valanciusand Smithies, Mol. Cell. Bio., 11: 1402-1408, 1991; Stacey et al., Mol.Cell. Bio., 14:1009-1016, 1994; Hasty et al., Nature, 350:243-246, 1995;Rubinstein et al., Nucl. Acid Res., 21:2613-2617,1993; Cibelli et al.,Science, 280:1256-1258, 1998. For guidance on recombinase excisionsystems, see, e.g., U.S. Pat. Nos. 5,626,159, 5,527,695, and 5,434,066.See also, Orban, P. C., et al., “Tissue-and Site-Specific DNARecombination in Transgenic Mice,” Proc. Natl. Acad. Sci. USA,89:6861-6865 (1992); O'Gorman, S., et al., “Recombinase-Mediated GeneActivation and Site-Specific Integration in Mammalian Cells,” Science,251:1351-1355 (1991); Sauer, B., et al., “Cre-stimulated recombinationat loxP-Containing DNA sequences placed into the mammalian genome,”Polynucleotides Research, 17(1):147-161 (1989); Gagneten, S. et al.(1997) Nucl. Acids Res. 25:3326-3331; Xiao and Weaver (1997) Nucl. AcidsRes. 25:2985-2991; Agah, R. et al. (1997) J. Clin. Invest. 100:169-179;Barlow, C. et al. (1997) Nucl. Acids Res. 25:2543-2545; Araki, K. et al.(1997) Nucl. Acids Res. 25:868-872; Mortensen, R. N. et al. (1992) Mol.Cell. Biol. 12:2391-2395 (G418 escalation method); Lakhlani, P. P. etal. (1997) Proc. Natl. Acad. Sci. USA 94:9950-9955 (“hit and run”);Westphal and Leder (1997) Curr. Biol. 7:530-533 (transposon-generated“knock-out” and “knock-in”); Templeton, N. S. et al. (1997) Gene Ther.4:700-709 (methods for efficient gene targeting, allowing for a highfrequency of homologous recombination events, e.g., without selectablemarkers); PCT International Publication WO 93/22443(functionally-disrupted).

[0137] A polynucleotide according to the present invention can beintroduced into any non-human animal, including a non-human mammal,mouse (Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1986), pig(Hammer et al., Nature, 315:343-345, 1985), sheep (Hammer et al.,Nature, 315:343-345, 1985), cattle, rat, or primate. See also, e.g.,Church, 1987, Trends in Biotech. 5:13-19; Clark et al., Trends inBiotech. 5:20-24, 1987); and DePamphilis et al., BioTechniques,6:662-680, 1988. Transgenic animals can be produced by the methodsdescribed in U.S. Pat. No. 5,994,618, and utilized for any of theutilities described therein.

[0138] Database

[0139] The present invention also relates to electronic forms ofpolynucleotides, polypeptides, etc., of the present invention, includingcomputer-readable medium (e.g., magnetic, optical, etc., stored in anysuitable format, such as flat files or hierarchical files) whichcomprise such sequences, or fragments thereof, e-commerce-related means,etc. Along these lines, the present invention relates to methods ofretrieving gene sequences from a computer-readable medium, comprising,one or more of the following steps in any effective order, e.g.,selecting a cell or gene expression profile, e.g., a profile thatspecifies that said gene is differentially expressed in, e.g., heart,prostate, and lung, and retrieving said differentially expressed genesequences, where the gene sequences consist of the genes represented bySEQ ID NOS 1 and 2.

[0140] A “gene expression profile” means the list of tissues, cells,etc., in which a defined gene is expressed (i.e, transcribed and/ortranslated). A “cell expression profile” means the genes which areexpressed in the particular cell type. The profile can be a list of thetissues in which the gene is expressed, but can include additionalinformation as well, including level of expression (e.g., a quantity ascompared or normalized to a control gene), and information on temporal(e.g., at what point in the cell-cycle or developmental program) andspatial expression. By the phrase “selecting a gene or cell expressionprofile,” it is meant that a user decides what type of gene or cellexpression pattern he is interested in retrieving, e.g., he may requirethat the gene is differentially expressed in a tissue, or he may requirethat the gene is not expressed in blood, but must be expressed inprostate. Any pattern of expression preferences may be selected. Theselecting can be performed by any effective method. In general,“selecting” refers to the process in which a user forms a query that isused to search a database of gene expression profiles. The step ofretrieving involves searching for results in a database that correspondto the query set forth in the selecting step. Any suitable algorithm canbe utilized to perform the search query, including algorithms that lookfor matches, or that perform optimization between query and data. Thedatabase is information that has been stored in an appropriate storagemedium, having a suitable computer-readable format. Once results areretrieved, they can be displayed in any suitable format, such as HTML.

[0141] For instance, the user may be interested in identifying genesthat are differentially expressed in prostate and heart. He may not carewhether small amounts of expression occur in other tissues, as long assuch genes are not expressed in peripheral blood lymphocytes. A query isformed by the user to retrieve the set of genes from the database havingthe desired gene or cell expression profile. Once the query is inputtedinto the system, a search algorithm is used to interrogate the database,and retrieve results.

[0142] Advertising, Licensing, etc., Methods

[0143] The present invention also relates to methods of advertising,licensing, selling, purchasing, brokering, etc., genes, polynucleotides,specific-binding partners, antibodies, etc., of the present invention.Methods can comprises, e.g., displaying a Tbx3-pr408 gene, Tbx3-pr408polypeptide, or antibody specific for Tbx3-pr408 in a printed orcomputer-readable medium (e.g., on the Web or Internet), accepting anoffer to purchase said gene, polypeptide, or antibody.

[0144] Other

[0145] A polynucleotide, probe, polypeptide, antibody, specific-bindingpartner, etc., according to the present invention can be isolated. Theterm “isolated” means that the material is in a form in which it is notfound in its original environment or in nature, e.g., more concentrated,more purified, separated from component, etc. An isolated polynucleotideincludes, e.g., a polynucleotide having the sequenced separated from thechromosomal DNA found in a living animal, e.g., as the complete gene, atranscript, or a cDNA. This polynucleotide can be part of a vector orinserted into a chromosome (by specific gene-targeting or by randomintegration at a position other than its normal position) and still beisolated in that it is not in a form that is found in its naturalenvironment. A polynucleotide, polypeptide, etc., of the presentinvention can also be substantially purified. By substantially purified,it is meant that polynucleotide or polypeptide is separated and isessentially free from other polynucleotides or polypeptides, i.e., thepolynucleotide or polypeptide is the primary and active constituent. Apolynucleotide can also be a recombinant molecule. By “recombinant,” itis meant that the polynucleotide is an arrangement or form which doesnot occur in nature. For instance, a recombinant molecule comprising apromoter sequence would not encompass the naturally-occurring gene, butwould include the promoter operably linked to a coding sequence notassociated with it in nature, e.g., a reporter gene, or a truncation ofthe normal coding sequence.

[0146] The term “marker” is used herein to indicate a means fordetecting or labeling a target. A marker can be a polynucleotide(usually referred to as a “probe”), polypeptide (e.g., an antibodyconjugated to a detectable label), PNA, or any effective material.

[0147] The topic headings set forth above are meant as guidance wherecertain information can be found in the application, but are notintended to be the only source in the application where information onsuch topic can be found. Reference materials

[0148] For other aspects of the polynucleotides, reference is made tostandard textbooks of molecular biology. See, e.g., Hames et al.,Polynucleotide Hybridization, IL Press, 1985; Davis et al., BasicMethods in Molecular Biology, Elsevir Sciences Publishing, Inc., NewYork, 1986; Sambrook et al., Molecular Cloning, CSH Press, 1989; Howe,Gene Cloning and Manipulation, Cambridge University Press, 1995; Ausubelet al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc.,1994-1998.

[0149] The preceding description, utilize the present invention to itsfullest extent. The preceding preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limiting theremainder of the disclosure in any way whatsoever. The entire disclosureof all applications, patents and publications, cited above and in thefigures are hereby incorporated by reference in their entirety.

1 5 1 3113 DNA Homo sapiens CDS (524)..(2695) 1 caccagcgag aaagagggagaggaagacag atagggggcg ggggaagaag aaaaagaaag 60 gtaaaaagtc ttctaggagaacctttcaca tttgcaacaa aagacctagg ggctggagag 120 agattcctgg gacgcagggctggagtgtct atttcgagct cagcggcagg gctcgggcgc 180 gagtcgagac cctgctcgctcctctcgctt ctgaaaccga cgttcaggag cggcttttta 240 aaaacgcaag gcacaaggacggtcacccgc gcgactatgt ttgctgattt ttcgccttgc 300 cctctttaaa agcggcctcccattccccaa aagacacttc ccctcctccc tttgaagtgc 360 attagttgtg atttctgcctccttttcttt tttctttctt ttttgttttg ctttttcccc 420 ccttttgaat tatgtgctgctgttaaacaa caacaaaaaa acaacaaaac acagcagctg 480 cggacttgtc cccggctggagcccagcgcc ccgcctggag tgg atg agc ctc tcc 535 Met Ser Leu Ser 1 atg agagat ccg gtc att cct ggg aca agc atg gcc tac cat ccg ttc 583 Met Arg AspPro Val Ile Pro Gly Thr Ser Met Ala Tyr His Pro Phe 5 10 15 20 cta cctcac cgg gcg ccg gac ttc gcc atg agc gcg gtg ctg ggt cac 631 Leu Pro HisArg Ala Pro Asp Phe Ala Met Ser Ala Val Leu Gly His 25 30 35 cag ccg ccgttc ttc ccc gcg ctg acg ctg cct ccc aac ggc gcg gcg 679 Gln Pro Pro PhePhe Pro Ala Leu Thr Leu Pro Pro Asn Gly Ala Ala 40 45 50 gcg ctc tcg ctgccg ggc gcc ctg gcc aag ccg atc atg gat caa ttg 727 Ala Leu Ser Leu ProGly Ala Leu Ala Lys Pro Ile Met Asp Gln Leu 55 60 65 gtg ggg gcg gcc gagacc ggc atc ccg ttc tcc tcc ctg ggg ccc cag 775 Val Gly Ala Ala Glu ThrGly Ile Pro Phe Ser Ser Leu Gly Pro Gln 70 75 80 gcg cat ctg agg cct ttgaag acc atg gag ccc gaa gaa gag gtg gag 823 Ala His Leu Arg Pro Leu LysThr Met Glu Pro Glu Glu Glu Val Glu 85 90 95 100 gac gac ccc aag gtg cacctg gag gct aaa gaa ctt tgg gat cag ttt 871 Asp Asp Pro Lys Val His LeuGlu Ala Lys Glu Leu Trp Asp Gln Phe 105 110 115 cac aag cgg ggc acc gagatg gtc att acc aag tcg gga agg cga atg 919 His Lys Arg Gly Thr Glu MetVal Ile Thr Lys Ser Gly Arg Arg Met 120 125 130 ttt cct cca ttt aaa gtgaga tgt tct ggg ctg gat aaa aaa gcc aaa 967 Phe Pro Pro Phe Lys Val ArgCys Ser Gly Leu Asp Lys Lys Ala Lys 135 140 145 tac att tta ttg atg gacatt ata gct gct gat gac tgt cgt tat aaa 1015 Tyr Ile Leu Leu Met Asp IleIle Ala Ala Asp Asp Cys Arg Tyr Lys 150 155 160 ttt cac aat tct cgg tggatg gtg gct ggt aag gcc gac ccc gaa atg 1063 Phe His Asn Ser Arg Trp MetVal Ala Gly Lys Ala Asp Pro Glu Met 165 170 175 180 cca aag agg atg tacatt cac ccg gac agc ccc gct act ggg gaa cag 1111 Pro Lys Arg Met Tyr IleHis Pro Asp Ser Pro Ala Thr Gly Glu Gln 185 190 195 tgg atg tcc aaa gtcgtc act ttc cac aaa ctg aaa ctc acc aac aac 1159 Trp Met Ser Lys Val ValThr Phe His Lys Leu Lys Leu Thr Asn Asn 200 205 210 att tca gac aaa catgga ttt act ata ttg aac tcc atg cac aaa tac 1207 Ile Ser Asp Lys His GlyPhe Thr Ile Leu Asn Ser Met His Lys Tyr 215 220 225 cag ccc cgg ttc cacatt gta aga gcc aat gac atc ttg aaa ctc cct 1255 Gln Pro Arg Phe His IleVal Arg Ala Asn Asp Ile Leu Lys Leu Pro 230 235 240 tat agt aca ttt cggaca tac ttg ttc ccc gaa act gaa ttc atc gct 1303 Tyr Ser Thr Phe Arg ThrTyr Leu Phe Pro Glu Thr Glu Phe Ile Ala 245 250 255 260 gtg act gca taccag aat gat aag ata acc cag tta aaa ata gac aac 1351 Val Thr Ala Tyr GlnAsn Asp Lys Ile Thr Gln Leu Lys Ile Asp Asn 265 270 275 aac cct ttt gcaaaa ggt ttc cgg gac act gga aat ggc cga aga gaa 1399 Asn Pro Phe Ala LysGly Phe Arg Asp Thr Gly Asn Gly Arg Arg Glu 280 285 290 aaa aga aaa cagctc acc ctg cag tcc atg agg gtg ttt gat gaa aga 1447 Lys Arg Lys Gln LeuThr Leu Gln Ser Met Arg Val Phe Asp Glu Arg 295 300 305 cac aaa aag gagaat ggg acc tct gat gag tcc tcc agt gaa caa gca 1495 His Lys Lys Glu AsnGly Thr Ser Asp Glu Ser Ser Ser Glu Gln Ala 310 315 320 gct ttc aac tgcttc gcc cag gct tct tct cca gcc gcc tcc act gta 1543 Ala Phe Asn Cys PheAla Gln Ala Ser Ser Pro Ala Ala Ser Thr Val 325 330 335 340 ggg aca tcgaac ctc aaa gat tta tgt ccc agc gag ggt gag agc gac 1591 Gly Thr Ser AsnLeu Lys Asp Leu Cys Pro Ser Glu Gly Glu Ser Asp 345 350 355 gcc gag gccgag agc aaa gag gag cat ggc ccc gag gcc tgc gac gcg 1639 Ala Glu Ala GluSer Lys Glu Glu His Gly Pro Glu Ala Cys Asp Ala 360 365 370 gcc aag atctcc acc acc acg tcg gag gag ccc tgc cgt gac aag ggc 1687 Ala Lys Ile SerThr Thr Thr Ser Glu Glu Pro Cys Arg Asp Lys Gly 375 380 385 agc ccc gcggtc aag gct cac ctt ttc gct gct gag cgg ccc cgg gac 1735 Ser Pro Ala ValLys Ala His Leu Phe Ala Ala Glu Arg Pro Arg Asp 390 395 400 agc ggg cggctg gac aaa gcg tcg ccc gac tca cgc cat agc ccc gcc 1783 Ser Gly Arg LeuAsp Lys Ala Ser Pro Asp Ser Arg His Ser Pro Ala 405 410 415 420 acc atctcg tcc agc act cgc ggc ctg ggc gcg gag gag cgc agg agc 1831 Thr Ile SerSer Ser Thr Arg Gly Leu Gly Ala Glu Glu Arg Arg Ser 425 430 435 ccg gttcgc gag ggc aca gcg ccg gcc aag gtg gaa gag gcg cgc gcg 1879 Pro Val ArgGlu Gly Thr Ala Pro Ala Lys Val Glu Glu Ala Arg Ala 440 445 450 ctc ccgggc aag gag gcc ttc gcg ccg ctc acg gtg cag acg gac gcg 1927 Leu Pro GlyLys Glu Ala Phe Ala Pro Leu Thr Val Gln Thr Asp Ala 455 460 465 gcc gccgcg cac ctg gcc cag ggc ccc ctg cct ggc ctc ggc ttc gcc 1975 Ala Ala AlaHis Leu Ala Gln Gly Pro Leu Pro Gly Leu Gly Phe Ala 470 475 480 ccg ggcctg gcg ggc caa cag ttc ttc aac ggg cac ccg ctc ttc ctg 2023 Pro Gly LeuAla Gly Gln Gln Phe Phe Asn Gly His Pro Leu Phe Leu 485 490 495 500 cacccc agc cag ttt gcc atg ggg ggc gcc ttc tcc agc atg gcg gcc 2071 His ProSer Gln Phe Ala Met Gly Gly Ala Phe Ser Ser Met Ala Ala 505 510 515 gctggc atg ggt ccc ctc ctg gcc acg gtt tct ggg gcc tcc acc ggt 2119 Ala GlyMet Gly Pro Leu Leu Ala Thr Val Ser Gly Ala Ser Thr Gly 520 525 530 gtctcg ggc ctg gat tcc acg gcc atg gcc tct gcc gct gcg gcg cag 2167 Val SerGly Leu Asp Ser Thr Ala Met Ala Ser Ala Ala Ala Ala Gln 535 540 545 ggactg tcc ggg gcg tcc gcg gcc acc ctg ccc ttc cac ctc cag cag 2215 Gly LeuSer Gly Ala Ser Ala Ala Thr Leu Pro Phe His Leu Gln Gln 550 555 560 cacgtc ctg gcc tct cag ggc ctg gcc atg tcc cct ttc gga agc ctg 2263 His ValLeu Ala Ser Gln Gly Leu Ala Met Ser Pro Phe Gly Ser Leu 565 570 575 580ttc cct tac ccc tac acg tac atg gcc gca gcg gcg gcc gcc tcc tct 2311 PhePro Tyr Pro Tyr Thr Tyr Met Ala Ala Ala Ala Ala Ala Ser Ser 585 590 595gcg gca gcc tcc agc tcg gtg cac cgc cac ccc ttc ctc aat ctg aac 2359 AlaAla Ala Ser Ser Ser Val His Arg His Pro Phe Leu Asn Leu Asn 600 605 610acc atg cgc ccg cgg ctg cgc tac agc ccc tac tcc atc ccg gtg ccg 2407 ThrMet Arg Pro Arg Leu Arg Tyr Ser Pro Tyr Ser Ile Pro Val Pro 615 620 625gtc ccg gac ggc agc agt ctg ctc acc acc gcc ctg ccc tcc atg gcg 2455 ValPro Asp Gly Ser Ser Leu Leu Thr Thr Ala Leu Pro Ser Met Ala 630 635 640gcg gcc gcg ggg ccc ctg gac ggc aaa gtc gcc gcc ctg gcc gcc agc 2503 AlaAla Ala Gly Pro Leu Asp Gly Lys Val Ala Ala Leu Ala Ala Ser 645 650 655660 ccg gcc tcg gtg gca gtg gac tcg ggc tct gaa ctc aac agc cgc tcc 2551Pro Ala Ser Val Ala Val Asp Ser Gly Ser Glu Leu Asn Ser Arg Ser 665 670675 tcc acg ctc tcc tcc agc tcc atg tcc ttg tcg ccc aaa ctc tgc gcg 2599Ser Thr Leu Ser Ser Ser Ser Met Ser Leu Ser Pro Lys Leu Cys Ala 680 685690 gag aaa gag gcg gcc acc agc gaa ctg cag agc atc cag cgg ttg gtt 2647Glu Lys Glu Ala Ala Thr Ser Glu Leu Gln Ser Ile Gln Arg Leu Val 695 700705 agc ggc ttg gaa gcc aag ccg gac agg tcc cgc agc gcg tcc ccg tag 2695Ser Gly Leu Glu Ala Lys Pro Asp Arg Ser Arg Ser Ala Ser Pro 710 715 720acccgtccca gacacgtctt ttcattccag tccagttcag gctgccgtgc actttgtcgg 2755atataaaata aaccacgggc ccgccatggc gttagccctt ccttttgcag ttgcgtctgg 2815gaaggggccc cggactccct cgagagaatg tgctagagac agcccctgtc ttcttggcgt 2875ggtttatatg tccgggatct ggatcagatt ctgggggctc agaaacgtcg gttgcattga 2935gctactgggg gtaggagttc caacatttat gtccagagca acttccagca aggctggtct 2995gggtctctgc ccaccaggcg gggaggtgtt caaagacatc tccctcagtg cggatttata 3055tatatatttt tccttcactg tgtcaagtgg aaacaaaaac aaaaaaaaaa aaaaaaaa 3113 2723 PRT Homo sapiens 2 Met Ser Leu Ser Met Arg Asp Pro Val Ile Pro GlyThr Ser Met Ala 1 5 10 15 Tyr His Pro Phe Leu Pro His Arg Ala Pro AspPhe Ala Met Ser Ala 20 25 30 Val Leu Gly His Gln Pro Pro Phe Phe Pro AlaLeu Thr Leu Pro Pro 35 40 45 Asn Gly Ala Ala Ala Leu Ser Leu Pro Gly AlaLeu Ala Lys Pro Ile 50 55 60 Met Asp Gln Leu Val Gly Ala Ala Glu Thr GlyIle Pro Phe Ser Ser 65 70 75 80 Leu Gly Pro Gln Ala His Leu Arg Pro LeuLys Thr Met Glu Pro Glu 85 90 95 Glu Glu Val Glu Asp Asp Pro Lys Val HisLeu Glu Ala Lys Glu Leu 100 105 110 Trp Asp Gln Phe His Lys Arg Gly ThrGlu Met Val Ile Thr Lys Ser 115 120 125 Gly Arg Arg Met Phe Pro Pro PheLys Val Arg Cys Ser Gly Leu Asp 130 135 140 Lys Lys Ala Lys Tyr Ile LeuLeu Met Asp Ile Ile Ala Ala Asp Asp 145 150 155 160 Cys Arg Tyr Lys PheHis Asn Ser Arg Trp Met Val Ala Gly Lys Ala 165 170 175 Asp Pro Glu MetPro Lys Arg Met Tyr Ile His Pro Asp Ser Pro Ala 180 185 190 Thr Gly GluGln Trp Met Ser Lys Val Val Thr Phe His Lys Leu Lys 195 200 205 Leu ThrAsn Asn Ile Ser Asp Lys His Gly Phe Thr Ile Leu Asn Ser 210 215 220 MetHis Lys Tyr Gln Pro Arg Phe His Ile Val Arg Ala Asn Asp Ile 225 230 235240 Leu Lys Leu Pro Tyr Ser Thr Phe Arg Thr Tyr Leu Phe Pro Glu Thr 245250 255 Glu Phe Ile Ala Val Thr Ala Tyr Gln Asn Asp Lys Ile Thr Gln Leu260 265 270 Lys Ile Asp Asn Asn Pro Phe Ala Lys Gly Phe Arg Asp Thr GlyAsn 275 280 285 Gly Arg Arg Glu Lys Arg Lys Gln Leu Thr Leu Gln Ser MetArg Val 290 295 300 Phe Asp Glu Arg His Lys Lys Glu Asn Gly Thr Ser AspGlu Ser Ser 305 310 315 320 Ser Glu Gln Ala Ala Phe Asn Cys Phe Ala GlnAla Ser Ser Pro Ala 325 330 335 Ala Ser Thr Val Gly Thr Ser Asn Leu LysAsp Leu Cys Pro Ser Glu 340 345 350 Gly Glu Ser Asp Ala Glu Ala Glu SerLys Glu Glu His Gly Pro Glu 355 360 365 Ala Cys Asp Ala Ala Lys Ile SerThr Thr Thr Ser Glu Glu Pro Cys 370 375 380 Arg Asp Lys Gly Ser Pro AlaVal Lys Ala His Leu Phe Ala Ala Glu 385 390 395 400 Arg Pro Arg Asp SerGly Arg Leu Asp Lys Ala Ser Pro Asp Ser Arg 405 410 415 His Ser Pro AlaThr Ile Ser Ser Ser Thr Arg Gly Leu Gly Ala Glu 420 425 430 Glu Arg ArgSer Pro Val Arg Glu Gly Thr Ala Pro Ala Lys Val Glu 435 440 445 Glu AlaArg Ala Leu Pro Gly Lys Glu Ala Phe Ala Pro Leu Thr Val 450 455 460 GlnThr Asp Ala Ala Ala Ala His Leu Ala Gln Gly Pro Leu Pro Gly 465 470 475480 Leu Gly Phe Ala Pro Gly Leu Ala Gly Gln Gln Phe Phe Asn Gly His 485490 495 Pro Leu Phe Leu His Pro Ser Gln Phe Ala Met Gly Gly Ala Phe Ser500 505 510 Ser Met Ala Ala Ala Gly Met Gly Pro Leu Leu Ala Thr Val SerGly 515 520 525 Ala Ser Thr Gly Val Ser Gly Leu Asp Ser Thr Ala Met AlaSer Ala 530 535 540 Ala Ala Ala Gln Gly Leu Ser Gly Ala Ser Ala Ala ThrLeu Pro Phe 545 550 555 560 His Leu Gln Gln His Val Leu Ala Ser Gln GlyLeu Ala Met Ser Pro 565 570 575 Phe Gly Ser Leu Phe Pro Tyr Pro Tyr ThrTyr Met Ala Ala Ala Ala 580 585 590 Ala Ala Ser Ser Ala Ala Ala Ser SerSer Val His Arg His Pro Phe 595 600 605 Leu Asn Leu Asn Thr Met Arg ProArg Leu Arg Tyr Ser Pro Tyr Ser 610 615 620 Ile Pro Val Pro Val Pro AspGly Ser Ser Leu Leu Thr Thr Ala Leu 625 630 635 640 Pro Ser Met Ala AlaAla Ala Gly Pro Leu Asp Gly Lys Val Ala Ala 645 650 655 Leu Ala Ala SerPro Ala Ser Val Ala Val Asp Ser Gly Ser Glu Leu 660 665 670 Asn Ser ArgSer Ser Thr Leu Ser Ser Ser Ser Met Ser Leu Ser Pro 675 680 685 Lys LeuCys Ala Glu Lys Glu Ala Ala Thr Ser Glu Leu Gln Ser Ile 690 695 700 GlnArg Leu Val Ser Gly Leu Glu Ala Lys Pro Asp Arg Ser Arg Ser 705 710 715720 Ala Ser Pro 3 722 PRT Homo sapiens 3 Met Ser Leu Ser Met Arg Asp ProVal Ile Pro Gly Thr Ser Met Ala 1 5 10 15 Tyr His Pro Phe Leu Pro HisArg Ala Pro Asp Phe Ala Met Ser Ala 20 25 30 Val Leu Gly His Gln Pro ProPhe Phe Pro Ala Leu Thr Leu Pro Pro 35 40 45 Asn Gly Ala Ala Ala Leu SerLeu Pro Gly Ala Leu Ala Lys Pro Ile 50 55 60 Met Asp Gln Leu Val Gly AlaAla Glu Thr Gly Ile Pro Phe Ser Ser 65 70 75 80 Leu Gly Pro Gln Ala HisLeu Arg Pro Leu Lys Thr Met Glu Pro Glu 85 90 95 Glu Glu Val Glu Asp AspPro Lys Val His Leu Glu Ala Lys Glu Leu 100 105 110 Trp Asp Gln Phe HisLys Arg Gly Thr Glu Met Val Ile Thr Lys Ser 115 120 125 Gly Arg Arg MetPhe Pro Pro Phe Lys Val Arg Cys Ser Gly Leu Asp 130 135 140 Lys Lys AlaLys Tyr Ile Leu Leu Met Asp Ile Ile Ala Ala Asp Asp 145 150 155 160 CysArg Tyr Lys Phe His Asn Ser Arg Trp Met Val Ala Gly Lys Ala 165 170 175Asp Pro Glu Met Pro Lys Arg Met Tyr Ile His Pro Asp Ser Pro Ala 180 185190 Thr Gly Glu Gln Trp Met Ser Lys Val Val Thr Phe His Lys Leu Lys 195200 205 Leu Thr Asn Asn Ile Ser Asp Lys His Gly Phe Thr Ile Leu Asn Ser210 215 220 Met His Lys Tyr Gln Pro Arg Phe His Ile Val Arg Ala Asn AspIle 225 230 235 240 Leu Lys Leu Pro Tyr Ser Thr Phe Arg Thr Tyr Leu PhePro Glu Thr 245 250 255 Glu Phe Ile Ala Val Thr Ala Tyr Gln Asn Asp LysIle Thr Gln Leu 260 265 270 Lys Ile Asp Asn Asn Pro Phe Ala Lys Gly PheArg Asp Thr Gly Asn 275 280 285 Gly Arg Arg Glu Lys Arg Lys Gln Leu ThrLeu Gln Ser Met Arg Val 290 295 300 Phe Asp Glu Arg His Lys Lys Glu AsnGly Thr Ser Asp Glu Ser Ser 305 310 315 320 Ser Glu Gln Ala Ala Phe AsnCys Phe Ala Gln Ala Ser Ser Pro Ala 325 330 335 Ala Ser Thr Val Gly ThrSer Asn Leu Lys Asp Leu Cys Pro Ser Glu 340 345 350 Gly Glu Ser Asp AlaGlu Ala Glu Ser Lys Glu Glu His Gly Pro Glu 355 360 365 Ala Cys Asp AlaAla Lys Ile Ser Thr Thr Thr Ser Glu Glu Pro Cys 370 375 380 Arg Asp LysGly Ser Pro Ala Val Lys Ala His Leu Phe Ala Ala Glu 385 390 395 400 ArgPro Arg Asp Ser Gly Arg Leu Asp Lys Ala Ser Pro Asp Ser Arg 405 410 415His Ser Pro Ala Thr Ile Ser Ser Ser Thr Arg Gly Leu Gly Ala Glu 420 425430 Glu Arg Arg Ser Pro Val Arg Glu Gly Thr Ala Pro Ala Lys Val Glu 435440 445 Glu Ala Arg Ala Leu Pro Gly Lys Glu Ala Phe Ala Pro Leu Thr Val450 455 460 Gln Thr Asp Ala Ala Ala Ala His Leu Ala Gln Gly Pro Leu ProGly 465 470 475 480 Leu Gly Phe Ala Pro Gly Leu Ala Gly Gln Gln Phe PheAsn Gly His 485 490 495 Pro Leu Phe Leu His Pro Ser Gln Phe Ala Met GlyGly Ala Phe Ser 500 505 510 Ser Met Ala Ala Ala Gly Met Gly Pro Leu LeuAla Thr Val Ser Gly 515 520 525 Ala Ser Thr Gly Val Ser Gly Leu Asp SerThr Ala Met Ala Ser Ala 530 535 540 Ala Ala Ala Gln Gly Leu Ser Gly AlaSer Ala Ala Thr Leu Pro Phe 545 550 555 560 His Leu Gln Gln His Val LeuAla Ser Gln Gly Leu Ala Met Ser Pro 565 570 575 Phe Gly Ser Leu Phe ProTyr Pro Tyr Thr Tyr Met Ala Ala Ala Ala 580 585 590 Ala Ala Ser Leu ArgGln Pro Gln Leu Arg Cys Thr Ala Pro Leu Leu 595 600 605 Asn Leu Asn ThrMet Arg Pro Arg Leu Arg Tyr Ser Pro Tyr Ser Ile 610 615 620 Pro Val ProVal Pro Asp Gly Ser Ser Leu Leu Thr Thr Ala Leu Pro 625 630 635 640 SerMet Ala Ala Ala Ala Gly Pro Leu Asp Gly Lys Ala Ala Ala Leu 645 650 655Ala Ala Ser Pro Ala Ser Val Ala Val Asp Ser Gly Ser Glu Pro Asn 660 665670 Ser Arg Ser Ser Thr Leu Ser Ser Ser Ser Met Ser Leu Ser Pro Lys 675680 685 Leu Cys Ala Glu Lys Glu Ala Ala Thr Ser Glu Leu Gln Ser Ile Gln690 695 700 Arg Leu Val Ser Gly Leu Glu Ala Lys Pro Asp Arg Ser Arg SerAla 705 710 715 720 Ser Pro 4 743 PRT Homo sapiens 4 Met Ser Leu Ser MetArg Asp Pro Val Ile Pro Gly Thr Ser Met Ala 1 5 10 15 Tyr His Pro PheLeu Pro His Arg Ala Pro Asp Phe Ala Met Ser Ala 20 25 30 Val Leu Gly HisGln Pro Pro Phe Phe Pro Ala Leu Thr Leu Pro Pro 35 40 45 Asn Gly Ala AlaAla Leu Ser Leu Pro Gly Ala Leu Ala Lys Pro Ile 50 55 60 Met Asp Gln LeuVal Gly Ala Ala Glu Thr Gly Ile Pro Phe Ser Ser 65 70 75 80 Leu Gly ProGln Ala His Leu Arg Pro Leu Lys Thr Met Glu Pro Glu 85 90 95 Glu Glu ValGlu Asp Asp Pro Lys Val His Leu Glu Ala Lys Glu Leu 100 105 110 Trp AspGln Phe His Lys Arg Gly Thr Glu Met Val Ile Thr Lys Ser 115 120 125 GlyArg Arg Met Phe Pro Pro Phe Lys Val Arg Cys Ser Gly Leu Asp 130 135 140Lys Lys Ala Lys Tyr Ile Leu Leu Met Asp Ile Ile Ala Ala Asp Asp 145 150155 160 Cys Arg Tyr Lys Phe His Asn Ser Arg Trp Met Val Ala Gly Lys Ala165 170 175 Asp Pro Glu Met Pro Lys Arg Met Tyr Ile His Pro Asp Ser ProAla 180 185 190 Thr Gly Glu Gln Trp Met Ser Lys Val Val Thr Phe His LysLeu Lys 195 200 205 Leu Thr Asn Asn Ile Ser Asp Lys His Gly Phe Thr LeuAla Phe Pro 210 215 220 Ser Asp His Ala Thr Trp Gln Gly Asn Tyr Ser PheGly Thr Gln Thr 225 230 235 240 Ile Leu Asn Ser Met His Lys Tyr Gln ProArg Phe His Ile Val Arg 245 250 255 Ala Asn Asp Ile Leu Lys Leu Pro TyrSer Thr Phe Arg Thr Tyr Leu 260 265 270 Phe Pro Glu Thr Glu Phe Ile AlaVal Thr Ala Tyr Gln Asn Asp Lys 275 280 285 Ile Thr Gln Leu Lys Ile AspAsn Asn Pro Phe Ala Lys Gly Phe Arg 290 295 300 Asp Thr Gly Asn Gly ArgArg Glu Lys Arg Lys Gln Leu Thr Leu Gln 305 310 315 320 Ser Met Arg ValPhe Asp Glu Arg His Lys Lys Glu Asn Gly Thr Ser 325 330 335 Asp Glu SerSer Ser Glu Gln Ala Ala Phe Asn Cys Phe Ala Gln Ala 340 345 350 Ser SerPro Ala Ala Ser Thr Val Gly Thr Ser Asn Leu Lys Asp Leu 355 360 365 CysPro Ser Glu Gly Glu Ser Asp Ala Glu Ala Glu Ser Lys Glu Glu 370 375 380His Gly Pro Glu Ala Cys Asp Ala Ala Lys Ile Ser Thr Thr Thr Ser 385 390395 400 Glu Glu Pro Cys Arg Asp Lys Gly Ser Pro Ala Val Lys Ala His Leu405 410 415 Phe Ala Ala Glu Arg Pro Arg Asp Ser Gly Arg Leu Asp Lys AlaSer 420 425 430 Pro Asp Ser Arg His Ser Pro Ala Thr Ile Ser Ser Ser ThrArg Gly 435 440 445 Leu Gly Ala Glu Glu Arg Arg Ser Pro Val Arg Glu GlyThr Ala Pro 450 455 460 Ala Lys Val Glu Glu Ala Arg Ala Leu Pro Gly LysGlu Ala Phe Ala 465 470 475 480 Pro Leu Thr Val Gln Thr Asp Ala Ala AlaAla His Leu Ala Gln Gly 485 490 495 Pro Leu Pro Gly Leu Gly Phe Ala ProGly Leu Ala Gly Gln Gln Phe 500 505 510 Phe Asn Gly His Pro Leu Phe LeuHis Pro Ser Gln Phe Ala Met Gly 515 520 525 Gly Ala Phe Ser Ser Met AlaAla Ala Gly Met Gly Pro Leu Leu Ala 530 535 540 Thr Val Ser Gly Ala SerThr Gly Val Ser Gly Leu Asp Ser Thr Ala 545 550 555 560 Met Ala Ser AlaAla Ala Ala Gln Gly Leu Ser Gly Ala Ser Ala Ala 565 570 575 Thr Leu ProPhe His Leu Gln Gln His Val Leu Ala Ser Gln Gly Leu 580 585 590 Ala MetSer Pro Phe Gly Ser Leu Phe Pro Tyr Pro Tyr Thr Tyr Met 595 600 605 AlaAla Ala Ala Ala Ala Ser Ser Ala Ala Ala Ser Ser Ser Val His 610 615 620Arg His Pro Phe Leu Asn Leu Asn Thr Met Arg Pro Arg Leu Arg Tyr 625 630635 640 Ser Pro Tyr Ser Ile Pro Val Pro Val Pro Asp Gly Ser Ser Leu Leu645 650 655 Thr Thr Ala Leu Pro Ser Met Ala Ala Ala Ala Gly Pro Leu AspGly 660 665 670 Lys Val Ala Ala Leu Ala Ala Ser Pro Ala Ser Val Ala ValAsp Ser 675 680 685 Gly Ser Glu Leu Asn Ser Arg Ser Ser Thr Leu Ser SerSer Ser Met 690 695 700 Ser Leu Ser Pro Lys Leu Cys Ala Glu Lys Glu AlaAla Thr Ser Glu 705 710 715 720 Leu Gln Ser Ile Gln Arg Leu Val Ser GlyLeu Glu Ala Lys Pro Asp 725 730 735 Arg Ser Arg Ser Ala Ser Pro 740 5596 PRT Homo sapiens 5 Met Ser Leu Ser Met Arg Asp Pro Val Ile Pro GlyThr Ser Met Ala 1 5 10 15 Tyr His Pro Phe Leu Pro His Arg Ala Pro AspPhe Ala Met Ser Ala 20 25 30 Val Leu Gly His Gln Pro Pro Phe Phe Pro AlaLeu Thr Leu Pro Pro 35 40 45 Asn Gly Ala Ala Ala Leu Ser Leu Pro Gly AlaLeu Ala Lys Pro Ile 50 55 60 Met Asp Gln Leu Val Gly Ala Ala Glu Thr GlyIle Pro Phe Ser Ser 65 70 75 80 Leu Gly Pro Gln Ala His Leu Arg Pro LeuLys Thr Met Glu Pro Glu 85 90 95 Glu Glu Val Glu Asp Asp Pro Lys Val HisLeu Glu Ala Lys Glu Leu 100 105 110 Trp Asp Gln Phe His Lys Arg Gly ThrGlu Met Val Ile Thr Lys Ser 115 120 125 Gly Arg Arg Met Phe Pro Pro PheLys Val Arg Cys Ser Gly Leu Asp 130 135 140 Lys Lys Ala Lys Tyr Ile LeuLeu Met Asp Ile Ile Ala Ala Asp Asp 145 150 155 160 Cys Arg Tyr Lys PheHis Asn Ser Arg Trp Met Val Ala Gly Lys Ala 165 170 175 Asp Pro Glu MetPro Lys Arg Met Tyr Ile His Pro Asp Ser Pro Ala 180 185 190 Thr Gly GluGln Trp Met Ser Lys Val Val Thr Phe His Lys Leu Lys 195 200 205 Leu ThrAsn Asn Ile Ser Asp Lys His Gly Phe Thr Leu Ala Phe Pro 210 215 220 SerAsp His Ala Thr Trp Gln Gly Asn Tyr Ser Phe Gly Thr Gln Thr 225 230 235240 Ile Leu Asn Ser Met His Lys Tyr Gln Pro Arg Phe His Ile Val Arg 245250 255 Ala Asn Asp Ile Leu Lys Leu Pro Tyr Ser Thr Phe Arg Thr Tyr Leu260 265 270 Phe Pro Glu Thr Glu Phe Ile Ala Val Thr Ala Tyr Gln Asn AspLys 275 280 285 Ile Thr Gln Leu Lys Ile Asp Asn Asn Pro Phe Ala Lys GlyPhe Arg 290 295 300 Asp Thr Gly Asn Gly Arg Arg Glu Lys Arg Gln Gln LeuThr Leu Gln 305 310 315 320 Ser Met Arg Val Phe Asp Glu Arg His Lys LysGlu Asn Gly Thr Ser 325 330 335 Asp Glu Ser Ser Ser Glu Gln Ala Ala PheAsn Cys Phe Ala Gln Ala 340 345 350 Ser Ser Pro Ala Ala Ser Thr Val GlyThr Ser Asn Leu Lys Asp Leu 355 360 365 Cys Pro Ser Glu Gly Glu Ser AspAla Glu Ala Glu Ser Lys Glu Glu 370 375 380 His Gly Pro Glu Ala Cys AspAla Ala Lys Ile Ser Thr Thr Thr Ser 385 390 395 400 Glu Glu Pro Cys ArgAsp Lys Gly Ser Pro Ala Val Lys Ala His Leu 405 410 415 Phe Ala Ala GluArg Pro Arg Asp Ser Gly Arg Leu Asp Lys Ala Ser 420 425 430 Pro Asp SerArg His Ser Pro Ala Thr Ile Ser Ser Ser Thr Arg Gly 435 440 445 Leu GlyAla Glu Glu Arg Arg Ser Pro Val Arg Glu Gly Thr Ala Pro 450 455 460 AlaLys Val Glu Glu Ala Arg Ala Leu Pro Gly Lys Glu Ala Phe Ala 465 470 475480 Pro Leu Thr Val Gln Thr Asp Ala Ala Arg Ser Ser Val His Arg His 485490 495 Pro Phe Arg Asn Leu Asn Thr Met Arg Pro Arg Leu Arg Tyr Ser Pro500 505 510 Tyr Ser Ile Pro Val Pro Val Pro Asp Gly Ser Ser Leu Leu ThrThr 515 520 525 Ala Leu Ala Ala Ser Pro Ala Ser Val Ala Val Asp Ser GlySer Glu 530 535 540 Leu Asn Ser Arg Ser Ser Thr Leu Ser Ser Ser Ser MetSer Leu Ser 545 550 555 560 Pro Lys Leu Cys Ala Glu Lys Glu Ala Ala ThrSer Glu Leu Gln Ser 565 570 575 Ile Gln Arg Leu Val Ser Gly Leu Glu AlaLys Pro Asp Arg Ser Arg 580 585 590 Ser Ala Ser Pro 595

1. An isolated Tbx3-pr408 polynucleotide which codes withoutinterruption for an amino acid sequence set forth in SEQ ID NO 2, or acomplement thereto.
 2. An isolated Tbx3-pr408 polynucleotide comprising,polynucleotide sequence having 97% or more sequence identity to thepolynucleotide sequence set forth in SEQ ID NO 1, which codes withoutinterruption for Tbx3-pr408, or a complement thereto.
 3. An isolatedpolynucleotide of claim 2, having 97% or more sequence identity to thepolynucleotide sequence set forth in SEQ ID NO
 1. 4. An isolatedpolynucleotide of claim 3 having transcriptional regulatory activity. 5.An isolated Tbx3-pr408 polypeptide comprising, the amino acid sequenceset forth in SEQ ID NO
 2. 6. An isolated polypeptide, selected from theamino acid sequence 596-608 of SEQ ID NO 2 which is specific forTbx3-Pr408.
 7. An antibody which is specific-for a polypeptide of claim5.
 8. An isolated polypeptide comprising an amino acid sequence having99% or more sequence identity to the amino acid sequence set forth inSEQ ID NO
 2. 9. A method of diagnosing a disorder in a subjectassociated with Tbx3-pr408, or determining a subject's susceptibility tosuch disease, comprising: assessing the expression of Tbx3-pr408 ofclaim 1 in a tissue sample from said subject.
 10. A method of claim 9,wherein assessing is: measuring expression levels of said gene,determining the genomic structure of said gene, determining the mRNAstructure of transcripts from said gene, or measuring the expressionlevels of polypeptide coded for by said gene.
 11. A method of claim 9,wherein the expression of Tbx3-pr408 and XM_(—)016321 are assessed. 12.A method of claim 9, wherein said assessing detecting is performed by:Northern blot analysis, polymerase chain reaction (PCR), reversetranscriptase PCR, RACE PCR, or in situ hybridization, and using apolynucleotide probe having a sequence selected from SEQ ID NO 1, 3, 4,or 5, effective specific fragments thereof, or complements thereto. 13.A method for identifying an agent that modulates the transcriptionalregulatory activity of Tbx3-pr408, comprising, contacting Tbx3-pr408 ofclaim 1, or a biologically-active fragment thereof which comprisesTbx3-pr408 specific sequence, with a test agent under conditionseffective for said test agent to modulate its transcriptional regulatoryactivity, and determining whether said test agent modulates saidTbx3-pr408.
 14. A method of claim 13, wherein said agent is an antibodyspecific for a polypeptide comprising amino acids 596-608, or apolypeptide comprising amino acids 215-225.
 15. A method of detectingTbx transcripts in a tissue sample, comprising assessing the expressionof a Tbx3-pr408 transcript of claim 3 in a tissue sample, and assessingthe expression of a Tbx3 transcript comprising a T box insertion.
 16. Amethod of claim 15, wherein assessing is: measuring expression levels ofsaid gene, determining the genomic structure of said gene, determiningthe mRNA structure of transcripts from said gene, or measuring theexpression levels of polypeptide coded for by said gene.
 17. A method ofclaim 15, wherein the expression of Tbx3-pr408 and XM_(—)016321 areassessed.
 18. A mammalian cell, comprising an exogenous polynucleotideof claim
 1. 19. A method of advertising Tbx3-pr4-8 for sale, commercialuse, or licensing, comprising, displaying in a computer-readable mediuma polynucleotide, or polypeptide sequence thereto, of claim 1, effectivespecific fragments thereof, or complements thereto.